Method of redirecting T cells to treat HIV infection

ABSTRACT

The present invention relates to compositions and methods for treating of a HIV infected mammal using a CD4 membrane-bound chimeric receptor or a HIV specific scFvs CARs. One aspect includes a modified T cell and pharmaceutical compositions comprising the modified cells for adoptive cell therapy and treating a disease or condition associated with HIV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to International Application No.PCT/US2016/053097, filed Sep. 22, 2016, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/222,132,filed Sep. 22, 2015 and U.S. Provisional Patent Application No.62/253,790, filed Nov. 11, 2015, which are incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NosAI104280, AI117950, and AR064220 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Despite the ability of antiretroviral therapy to minimize humanimmunodeficiency virus type (HIV) replication and increase the durationand quality of patients' lives, the health consequences and financialburden associated with the lifelong treatment regimen render a permanentcure highly attractive. Although T cells play an important role incontrolling virus replication, they are themselves targets ofHIV-mediated destruction.

Restoration of CD4 T cell activity, whether by immune augmentation or byprotection from deletion, is a critical factor to enable long-termcontrol of HIV replication in the absence of highly activeantiretroviral therapy (HAART). Attempts to manufacture T cells astherapeutic agents to treat HW have been ongoing for over two decades. Tcells can be engineered to express a synthetic immunoreceptor comprisedof an extracellular targeted antibody and intracellular signalingdomain, known as chimeric antigen receptor (CAR). This new area ofresearch, referred to as adoptive T cell therapy, has recently undergonemany technological advances. Importantly, T-cell therapy approaches havethe potential to protect helper CD4 T cells and equip them with directantiviral functions, which may be critical for improving HIV-specificcytotoxicity and achieving control over HIV replication in the absenceof antiretroviral therapy. While major advances have already been madein the field of T cell engineering for adoptive therapy, includingdemonstrations of safety and feasibility, no clinical trial has resultedin durable and consistent control over HIV-replication in the absence ofHAART.

In contrast to a vaccine approach, which relies on the production andpriming of HIV-specific lymphocytes within a patient's own body,adoptive T-cell therapy provides an opportunity to customize thetherapeutic T cells prior to administration. Thus, despite theunsuccessful therapeutic attempts using direct genetic manipulation of Tcells, it is clear that adoptive cellular therapies could facilitate afunctional cure by generating HIV-resistant cells, redirectingHIV-specific immune responses, or a combination of the two strategies.However, at present, it is unclear how to best engineer T cells so thatsustained control over HIV replication can be achieved in the absence ofantiretroviral therapy.

There is a great need in the art for more effective T-cellgene-engineering and gene-editing strategies that inhibit HIVreplication and provide a gene therapy-mediated functional cure. Thisinvention addresses this need.

SUMMARY OF THE INVENTION

As described herein, the present invention relates to compositions andmethods for treating of a HIV infected mammal using a CD4 membrane-boundchimeric receptor or a HIV specific single chain variable fragments(scFvs) CARs.

In one aspect, the invention includes an isolated nucleic acid sequenceencoding a membrane-bound chimeric receptor. The isolated nucleic acidsequence comprises a CD4 extracellular domain, a transmembrane domain,and a signaling domain, wherein the CD4 extracellular domain is capableof recognizing and binding a HIV infected cell.

In another aspect, the invention includes an isolated amino acidsequence encoding a membrane-bound chimeric receptor. The isolated aminoacid sequence comprises a CD4 extracellular domain, a transmembranedomain, and a signaling domain, wherein the CD4 extracellular domain iscapable of recognizing and binding a HIV infected cell.

In some embodiments, the CD4 extracellular domain is encoded by anucleic acid sequence comprising SEQ ID NO: 3 or 64. In someembodiments, the CD4 extracellular domain comprises the amino acidsequence SEQ ID NO: 46. In some other embodiments, the CD4 extracellulardomain specifically binds to the HIV envelope (Env) glycoprotein.

In some embodiments, the transmembrane domain comprises a CD8alpha hingeencoded by nucleic acid sequence SEQ ID NO: 4 or 65 and a transmembranedomain comprising at least one domain encoded by a nucleic acid sequencecomprising one selected from the group consisting of SEQ ID NOs: 5 and8. In other embodiments, the transmembrane domain comprises thetransmembrane domain comprises a CD8alpha hinge of SEQ ID NO: 47, and atransmembrane domain comprising at least one amino acid sequenceselected from the group consisting of SEQ ID NOs: 48 and 49.

In other embodiments, the signaling domain comprises a CD3zeta signalingdomain encoded by a nucleic acid sequence comprising SEQ ID NO: 6 or 68.In other embodiments, the signaling domain comprises a CD3zeta signalingdomain comprising SEQ ID NO: 51.

In yet other embodiments, the isolated nucleic acid or amino acidsequence of the invention further comprises a costimulatory signalingregion.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the costimulatory signaling regioncomprises an intracellular domain of a costimulatory molecule selectedfrom the group consisting of alpha, beta or zeta chain of a TCR zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, or CD66d, a costimulatory signaling region from CD27,CD28, 4-1BB (CD137), DAP12, OX9, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83, and anycombination thereof. In some embodiments, the costimulatory signalingregion is CD28 and is encoded by a nucleic acid sequence comprising SEQID NO: 9 or 67. In other embodiments, the costimulatory signaling regionis CD28 and comprises SEQ ID NO: 49.

In one aspect, the invention includes a vector comprising an isolatednucleic acid sequence encoding a membrane-bound chimeric receptor. Theisolated nucleic acid sequence of the vector comprises a CD4extracellular domain, a transmembrane domain, and a signaling domain,wherein the CD4 extracellular domain is capable of recognizing andbinding a HIV infected cell. In some embodiments the nucleic acidsequence of the vector comprises at least one from the group consistingof SEQ ID NOs: 1, 7, 62 and 63.

In another aspect, the invention includes a vector comprising amembrane-bound chimeric receptor. The membrane-bound chimeric receptorof the vector comprises a CD4 extracellular domain, a transmembranedomain, and a signaling domain, wherein the CD4 extracellular domain iscapable of recognizing and binding a HIV infected cell, wherein thevector comprises at least one amino acid sequence selected from thegroup consisting of SEQ ID NOs: 44 and 45. In some embodiments, thevector comprises an EFu promoter.

In yet another aspect, the invention includes an isolated nucleic acidsequence encoding a chimeric antigen receptor (CAR). The isolatednucleic acid sequence encoding the CAR of the invention comprises aHIV-specific binding domain, a transmembrane domain, a costimulatorysignaling region, and a signaling domain, wherein the HIV binding domaincomprises an anti-HIV antibody or a fragment thereof.

In some embodiments, the HIV-specific binding domain comprises a heavyand light chain. In other embodiments, the HIV-specific binding domainis a human antibody, a humanized antibody, and a fragment thereof. Inother embodiments, the antibody or a fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a Fvfragment, and a single chain Fv (scFv). In yet other embodiments, thescFv comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 13, 17, 21, 25, 29, 57-60 and 61. In further embodiments,the scFv is encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 16, 20, 24, 28, 75-78 and 79. In stillfurther embodiments, the HIV-specific binding domain specifically bindsto the surface of HIV infected cells, or HIV virions.

In some embodiments, the costimulatory signaling region of the CAR ofthe invention comprises an intracellular domain of a costimulatorymolecule selected from the group consisting of TCR, CD3 zeta, CD3 gamma,CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc EpsilonRib), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, T cell receptor (TCR),CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, a KIRfamily protein, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127,CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha,ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-likereceptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, andany combination thereof.

In some embodiments, the signaling domain of the CAR of the inventioncomprises a CD3zeta signaling domain encoded by nucleic acid sequenceSEQ ID NO: 6 or 68.

In some embodiments, the CAR is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 10, 14, 18, 22, 26,34, 70-73, and 74. In other embodiments, the CAR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 11, 15, 19,23, 27, 35, 52-55, and 56.

In one aspect, the invention includes a modified cell comprising eitheran isolated nucleic acid sequence that comprises a CD4 extracellulardomain, a transmembrane domain, and a signaling domain, wherein the CD4extracellular domain is capable of recognizing and binding a HIVinfected cell; or an isolated nucleic acid sequence encoding a CAR thatcomprises a HIV-specific binding domain, a transmembrane domain, acostimulatory signaling region, and a signaling domain, wherein the HIVbinding domain comprises an anti-HIV antibody or a fragment thereof.

In some embodiments, the modified cell of this invention cell isselected from the group consisting of a T cell, a natural killer (NK)cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell. In otherembodiments, the nucleic acid sequence of the modified cell is selectedfrom the group consisting of a DNA and an mRNA. In yet otherembodiments, the nucleic acid sequence is introduced into the cell by atleast one procedure selected from the group consisting ofelectroporation, usage of a lentivirus, usage of a retrovirus and achemical-based transfection.

In another aspect, the invention includes a composition comprising themodified cell of the invention as listed above herein.

In another aspect, the invention includes the use of the modified cellof this invention in the manufacture of a medicament for the treatmentof HIV infection in a subject in need thereof.

In yet another aspect, the invention includes a pharmaceuticalcomposition comprising the modified cell of the invention and apharmaceutically acceptable carrier.

In a further aspect, the invention includes a method for stimulating acellular immune response in a HIV infected mammal. The method comprisesadministering to the mammal an effective amount of the modified cell ofthe invention as listed above herein.

In yet a further aspect, the invention includes a method of treating aHIV infected mammal. The method comprises administering to the mammalthe modified cell of the invention as listed above herein. In someembodiments, the modified cell is autologous to the mammal. In otherembodiments, the method of the invention further comprises administeringantiretroviral therapy (HAART) the mammal. In yet other embodiments, themodified cell and the HAART are co-administered to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments, which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1B are series of graphs demonstrating that CD4 CAR is over100-fold more potent than HIV-specific elite controller TCR in vitro.FIG. 1A: CD4 and CD8 T cells were obtained from an HLA-B57+ normaldonor. CD4 T cells were infected with HIV Bal and, 24 hours later,non-transduced CD8 T cells (NTD), CD8 T cells expressing a HLA-B57restricted TCR specific for KAFSPEVIPMF-B57-KF11 (KF11), or CD8 T cellsexpressing an EF1α-CD8αTM CD4 CAR construct (CD4z), both expressed underthe EF1a promoter, were mixed in at the indicated effector to targetratios. Transduction efficiencies were normalized to 40% prior toco-culture. After 6 days of co-culture, Gag p24 and CD4 staining isshown for CD8 negative T cells. CD4 membrane-bound chimeric receptor(also referred to as CD4 zeta or CD4 zeta construct) controls NL4-3HIV-1 replication better than the HLA-B*57 restricted KF11 elitecontroller TCR. The KF11 TCR is associated with better control overHIV-1 replication in patients and is one of the most potentpatient-derived TCRs against HIV-1. This represents the best controlover HIV-1 that can be achieved with a TCR-based strategy. In contrastto KF11 TCR, the CD4 zeta was able to completely control HIV-1replication at all Effector Cell to Target Cell (E:T) ratios shown. FIG.1B: Summary data for a single experiment performed in triplicate, gatingon the CD8 negative cells. Error bars indicate standard error of themean (SEM). These data are representative of 3 independent experiments.

FIGS. 2A-2G are series of schematic representations and graphs depictingthat promoter and transmembrane changes improve CD4 membrane-boundchimeric receptor efficacy and increase control over HIV-1. FIG. 2A:schematic of the new, improved CD4 membrane-bound chimeric receptorconstruct (top) with the CD8α transmembrane (TM) and EF1α promotercompared to the construct that entered clinical trials (bottom) thatcontained the PGK promoter and the CD4 TM domains. FIG. 2B: Surfaceexpression of the CD4 zeta in transduced CD8s. Compared to theretroviral vector-based construct that entered clinical trials (farright), the same construct was expressed at a higher level when alentiviral vector was used (second from left). This expression wasincreased further when the EF1α promoter was substituted into thelentiviral vector (second from left). FIG. 2C: Experimental timeline forall of the data shown in the presentation. Healthy donor CD4 and CD8 Tlymphocytes were stimulated with αCD3/CD28 coated beads and 100-300IU/ml IL-2. After 24 hours the CD8s were transduced with lentivirus, oron days 3 and 5 transduced with retrovirus. After 5 days the beads wereremoved, and 48 hours later the CD4 T cells were infected. 24 hoursafter infecting the CD4 T cells, the CD8s were co-cultured in varyingE:T ratios. The cultures were fed with media and IL-2 and stained forCD4, CD8, and intracellular p24 every other day until HIV-1 replicationreached maximum replication capacity in the NTD wells (typically arounddays 10-12). FIGS. 2D-2E: Intracellular p24 stain 8 days followinginfection of the CD4 T cells with HIV-1 Bal. FIG. 2D, gating on the CD4T cells and FIG. 2E, gating on the CD8 T cells. The new, improved CD4zeta construct with the lentiviral vector, EF1α promoter, and CD8αtransmembrane domain controlled HIV-1 at much lower E:T ratios (untilroughly 1:100) than the constructs with the PGK promoter and CD4transmembrane domains (loss of control at 1:5 and 1:10). Although ahigher expression of the lentivirus PGK CD4 TM construct was observedwhen compared to the retrovirus PGK CD4 TM construct, a small benefitwas seen in terms of control over HIV-1 replication. Importantly, higherexpression of the EF1α CD8 TM construct did not promote higher rates ofinfection, as the CD8s remained uninfected until approximately the 1:100ratio whereas the PGK CD4 TM⁺ CD8s became infected at lower E:T ratios.FIG. 2F: Summary data for a single experiment performed in triplicate,gating on the CD8 negative cells. Error bars indicate standard error ofthe mean (SEM). FIG. 2G: Measurement of levels of intracellular p24 inCD8 negative T cells over the time course of an experiment. Each graphrepresents a different E:T ratio. These data are representative of threeindependent experiments.

FIGS. 3A-3E are series of schematic representations and graphs depictingthat EF1α promoter and CD8α transmembrane change is a criticalmodification to improve CAR expression and control of HIV-1 replication.FIG. 3A: schematic of the four lentiviral vector chimeric receptorconstructs that were generated to compare how changing the promoter andtransmembrane components each individually impacted control over HIV-1replication. Top construct refers to what was used in clinical trialsbut is now inserted into a lentiviral vector.

FIG. 3B: CD4 CAR expression on CD8 T cells 8 days after activation.Primary human CD8 T cells were activated with αCD3/αCD28 coated beadsand were either left non-transduced (NTD) or transduced with theindicated lentiviral vectors. After 8 days of culture, CAR expressionwas measured by CD4 staining. Median fluorescence intensity (MFI) ofeach construct is indicated on each graph. FIG. 3C: Intracellular p24stain 8 days following infection of the CD4 T cells with HIV-1 Bal. Leftset of columns was gated on the CD4 T cells and right set of columns wasgated on the CD8 T cells. As seen previously, the EF1α CD8TM constructcontrolled HIV-1 at the lowest E:T ratios. Simply increasing expressionof the CD4 TM with the EF1α promoter (middle column) improved controlover HIV-1 replication, but the combination of increased expression withthe EF1α promoter and CD8α transmembrane substitution produced the bestcontrol over HIV-1. CD8α TM appeared to reduce infection of the CD8 Tcells engineered with a chimeric receptor (right set of columns),particularly at the 1:50 ratio for the EF1α constructs and the 1:25ratio for the PGK constructs. FIG. 3D: Summary data for a singleexperiment performed in triplicate, gating on the CD8 negative cells.Error bars indicate standard error of the mean (SEM). FIG. 3E: Thelevels of intracellular p24 in CD8 negative T cells over the time courseof an experiment. Each graph represents a different E:T ratio. Thesedata are representative of three independent experiments.

FIGS. 4A-4C are a series of table and graphs demonstrating that bothsingle chain variable fragment (scFv) broadly neutralizing antibody(bnAb)-based CARs and CD4 membrane-bound chimeric receptor can controlHIV-1 infection. FIG. 4A: A panel of single-chain variable fragment(scFv) CARs adapted from HIV-1 specific bnAbs was generated under thehypothesis that these potent and broadly neutralizing HIV-specificantibodies would provide an alternate means of targeting HIV-infectedcells. A panel of scFvs (see table in FIG. 4A) that ranged in theirHIV-1 binding breadth and neutralization potency were cloned into thesame EF1α-CD8α TM-zeta construct backbone as the CD4 membrane-boundchimeric receptor and assessed for their ability to control HIV-1replication. Two weeks following activation, these T cells were mixedwith Cr51 labeled K562 target cells expressing HIV-1 YU2 GP160 at theindicated effector to target ratios. Specific lysis of the targets isplotted. Data plotted shows the average of three independentexperiments. Cr51 release killing assay from a 4 hour co-culture showedthat CD8 T cells expressing any of the scFv CARs or the CD4membrane-bound chimeric receptor, but not non-transduced (NTD) T cells,lysed Env-expressing target cells. This indicates the scFv CARs wereexpressed with proper folding/conformation and able to bind Env. FIG.4B: Primary human CD8 T cells were activated with αCD3/αCD28 coatedbeads and were either left non-transduced (NTD) or transduced withEF1α-CD8α TM lentiviral vectors encoding HIV-specific CARs derived fromthe VRC01, 3BNC60, PG9, PGT128, or PGDM1400 antibodies, or the CD4 CAR.Two weeks post activation, the CD8 T cells were co-cultured for 6 hoursat a 1:1 ratio with K562 cells expressing HIV-1 YU2 GP160 andintracellular IFNγ and MIP-1β production was measured. Transductionefficiencies were normalized to 60% prior to co-culture. FIG. 4C: Usingthe experimental design summarized in FIG. 2C, the HIV-specific CARswere tested for their ability to control HIV-1 replication in primaryhuman CD4 T cells. Transduction efficiencies were normalized to 70%prior to co-culture. Intracellular Gag p24 and CD4 stain 9 daysfollowing infection of the CD4 T cells with HIV-1 Bal, CD4 T cells areshown. Both the CD4 membrane-bound chimeric receptor and the scFv CARsdemonstrated lysis of Env target cells relative to NTD controls up to aE:T ratio of 1:50-1:200, which is superior to the activity of the KF11TCR shown in FIG. 1A, which demonstrates efficacy up to a 1:25 E:Tratio. Error bars indicate standard error of the mean (SEM). Data arerepresentative of three independent experiments.

FIGS. 5A-5B are series of graphs showing that CD28 costimulationpromotes control over HIV-1 Bal and that CD28 and 4-1BB costimulationhave opposing effects on the control of HIV-1 replication in vitro. FIG.5A: Intracellular p24 stain 10 days following infection of the CD4 Tcells with HIV-1 Bal, CD4 T cells is shown. A panel of costimulatorydomains that have been utilized in common CAR designs was cloned intothe CD4 membrane-bound chimeric receptor backbone to determine theeffects of costimulation on control over HIV-1 in vitro. The onlymembrane-bound chimeric receptor that consistently controlled HIV-1 aswell or better than CD4 zeta construct was the CD4 CD28 zeta construct.In contrast, the addition of 4-1BB, ICOS, and CD27 impaired control overHIV-1, and OX40 and a CD28-4-1BBzeta combination appeared to have littleeffect. FIG. 5B: Summary data for a single experiment performed intriplicate, gated on the CD8 negative T cells. Error bars indicatestandard error of the mean (SEM). Data are representative of threeindependent experiments.

FIG. 6 is the nucleotide sequence of CD4 membrane-bound chimericreceptor (CD4 zeta construct; SEQ ID NO: 1). This sequence has beenannotated in red for the section related to the EF1α promoter (SEQ IDNO: 2), in yellow for the CD4 extracellular domain (SEQ ID NO: 3), ingreen for the CD8α extracellular hinge (SEQ ID NO: 4), in Turquoise forthe CD8α transmembrane domain (SEQ ID NO: 5) and in pink for the CD3zeta (SEQ ID NO: 6).

FIG. 7 is the nucleotide sequence of CD4 CD28 membrane-bound chimericreceptor (CD4 CD28 zeta construct; SEQ ID NO: 7). This sequence has beenannotated in red for the section related to the EF1α promoter, in yellowfor the CD4 extracellular domain, in green for the CD8α extracellularhinge, in Turquoise for the CD28 transmembrane domain (SEQ ID NO: 8), inblue for the CD28 costimulatory domain (SEQ ID NO: 9) and in pink forthe CD3 zeta.

FIG. 8 is the listing of the nucleotide and amino acid (aa) sequences offive antibody based CAR constructs: PGDM1400 killer cellimmunoglobulin-like receptor (KIR) nucleotide and aa sequences (SEQ IDNOs: 10 and 11, respectively), PGDM1400 scFv nucleotide and aa sequences(SEQ ID NOs: 12 and 13, respectively), PGT128 KIR nucleotide and aasequences (SEQ ID NOs: 14 and 15, respectively), PGT128 scFv nucleotideand aa sequences (SEQ ID NOs: 16 and 17, respectively), VRC01 KIRnucleotide and aa sequences (SEQ ID NOs: 18 and 19, respectively), VRC01scFv nucleotide and aa sequences (SEQ ID NOs: 20 and 21, respectively),3BNC60-KIR nucleotide and aa sequences (SEQ ID NOs: 22 and 23,respectively), 3BNC60 scFv nucleotide and aa sequences (SEQ ID NOs: 24and 25, respectively), VRC01 c-mut-KIR nucleotide and aa sequences (SEQID NOs: 26 and 27, respectively), VRC01 c-mut scFv nucleotide and aasequences (SEQ ID NOs: 28 and 29, respectively), CD4 Dap12-KIRS2nucleotide and aa sequences (SEQ ID NOs: 30 and 31, respectively),CD4-KIR nucleotide and aa sequences (SEQ ID NOs: 32 and 33,respectively), and VRC01 IgG4 bbz nucleotide and aa sequences (SEQ IDNOs: 34 and 35, respectively).

FIGS. 9A-9C are a series of graphs and histograms demonstrating thatscFv based CAR's (scFv-CARs) cytotoxicity is specific and potent. VRC01CARs showed specific cytotoxicity against target cells expressing HIVYU-2 strain (FIG. 9A) but not cells expressing CD19 (FIG. 9B);cytotoxicity can be increased by using a killer cell immunoglobulin-likereceptor (KIR) cytoplasmic domain (FIG. 9A). Killing efficacy correlatedwith interferon gamma (IFNg) release by CAR T cells (FIG. 9C). Theduration of the ⁵¹Cr release assay was 4 hours. The KIR constructconsisted of DAP12 and the scFv linked to a KIRS2 transmembrane domain.The G4-bbz construct consisted of the scFv linked to an IgG4 hinge, aCD8 transmembrane domain and 4-1BB and CD3 zeta intracellular tail.bn01=VRC01; NTD=non-transduced control T cells.

FIGS. 10A-10C are a series of graphs and histograms depicting anotherinstance of cell specific cytotoxicity for scFv-CARs. 3BNC60 andVRC01c-CARs showed specific lysis of K562 cells expressing HIV-1envelope gp120/41 (YU-2 isolate) (FIG. 10A), but not CD19 expressingK562 cells (FIG. 10B). As shown previously in FIG. 9C, specific IFNgammaproduction by CAR T cells correlated with killing efficacy (FIG. 10C).3BNC60 relates to another CD4 binding site broadly neutralizingantibody. VRC01-c is a derivative of VRC01, which lacks a disulfide bondbetween HCDR3 and HCDR1 and may therefore show a greater percentage ofcorrectly folded scFvs compared to the original VRC01 scFv that involvesan additional, non-canonical disulfide-bond. For 3BNC60 and VRC01-c,there was no difference detectable in terms of killing between the KIRand the IgG4-bbz (also referred to as G4) construct.

FIGS. 11A-11C are series of graph demonstrating that CD4 CARsspecifically respond to Env+ cells and not MHC class II+ cells. FIG.11A: Primary human CD8 T cells were activated with αCD3/αCD28 coatedbeads and were either left non-transduced (NTD) or transduced withEF1α-CD8α TM lentiviral vectors encoding CD4 CARs expressing theCD3-zeta signaling domain, alone or in combination with CD28 or 4-1BBcostimulatory domains. Two weeks post activation, the CD8 T cells wereco-cultured for 6 hours at a 1:1 ratio with unmodified K562 cells, K562cells expressing high levels of HLA-DR (see FIG. 14), or K562 cellsexpressing HIV-1 YU2 GP160. Intracellular IFNγ and MIP-1β expression isshown on the left, and intracellular IL-2 expression and CD107a surfacemobilization is shown on the right. FIG. 11B: A co-culture assay wasdesigned to demonstrate that CD4 CAR+ CD8 T cells do not kill MHC classII-expressing target cells. Briefly, NTD or CD4 28z CAR transduced CD8 Tcells from FIG. 6A were co-cultured with K562 cells expressing HLA-A2and GFP as well as K562 expressing HLA-DR*0401 and mCherry at a 1:1:1ratio. Flow cytometry measuring GFP and mCherry expression was performedimmediately after mixing (0 hr) and after 3 days of co-culture (72 hr).FIG. 11C: Summary data for a single experiment performed in triplicate,measuring the ratio of HLA-A2/GFP-expressing cells toHLA-DR*0401/mCherry-expressing cells after 24, 48, and 72 hours ofculture. Error bars indicate standard error of the mean (SEM). Data arerepresentative of three independent experiments.

FIGS. 12A-12F are series of graphs depicting T cells expressingoptimized CD4 CAR control HIV-1 replication and expanded to much greaterlevels in vivo than the original CD4 CAR. Cohorts of 7 NSG (NOD-scidIL2Rgnull) mice were infused with 8 million CD4 T cells and 2 millionCD8 T cells. CD8 T cells were either left non-transduced (NTD),transduced with optimized (EF1α-CD8α TM, lentiviral vector) CD4-zetaCARs containing either 4-1BB or CD28 intracellular costimulatorydomains, or the clinical trial (MMLV-based, PGK-CD4TM) CD4 CAR, denotedas NTD, BBz, 28z, and CD4z, respectively. CD8 T cell transductionefficiencies were normalized to 50% prior to injection into mice. Threeweeks post injection, engraftment was measured to determine (FIG. 12A)baseline peripheral CD4 T cell counts and (FIG. 12C) CAR+ CD8 T cellcounts. Two days later mice were infected with HIV-1 Bal via tail veininjection. 22 Days post infection, (FIG. 12B) endpoint peripheral CD4 Tcell counts and (FIG. 12D) CAR+ CD8 T cell counts were obtained. (FIG.12E) Seven and (FIG. 12F) eighteen days post infection the copies ofplasma HIV RNA were measured. Mann Whitney Test was used to determinestatistical significance (p values: ns>0.05, *<0.05, **<0.01,***<0.0001).

FIGS. 13A-13C are series of graphs showing that CCR5-ZFN modified CD4CAR CD8 T cells are enriched in vivo. In the NSG mouse experimentaldesign described in FIG. 7, four additional cohorts were added in whichthe CD8 T cells were electroporated with CCR5 ZFN RNA 48 hours beforeactivation and CD4 CAR transduction, or left non-transduced (NTD). FIG.13A: After 22 days of HIV infection, splenic CD8 T cells were isolatedfrom HIV-infected mice and analyzed for CCR5 disruption frequency. FIG.13B: The ratio of CD4 CAR-expressing CD8 T cells to nontransduced CD8 Tcells was determined by staining for human CD4 and CD8 and performingflow cytometry on peripheral blood isolated 22 days post HIV or mockinfection. Flow cytometry plots are shown in FIG. 15. FIG. 13C: Eighteendays post infection, the copies of plasma HIV RNA were measured inHIV-infected mice, as in FIG. 12F, but with the four ZFN-treated groupsincorporated. Significance was detected using a Mann Whitney Test (pvalues: ns>0.05, *<0.05, **<0.01, ***<0.0001).

FIG. 14 is a graph depicting high levels of MHC class II expression onHLA-DR-transduced K562 cells. To confirm high expression of MHC class IIon the HLA-DR*0401-transduced K562 cells, HLA-DR expression was measuredby flow cytometry. As a gating control, K562 cells that had beentransduced with HLA-A2 were stained, as well as the high MHC class IIexpressing Raji B cells. A histogram overlaying the three cellpopulations is depicted.

FIG. 15 is a series of graphs depicting that 4-1BB costimulatory domainresults in greater T cell persistence in the absence of antigen. After22 days of mock infection, peripheral blood cells or splenic cells fromBBz or 28z transduced, ZFN-treated mice cohorts were stained for CARexpression with αCD4 and αCD8 antibodies. The top panel shows peripheralblood and the bottom panel shows splenic cells. One representative flowplot is shown in all cases from an HIV-infected mouse.

FIGS. 16A-16B are series of graphs demonstrating that CCR5 ZFN treatmentdoes not improve the ability of CD4 CAR CD8 T cells to protect CD4 Tcell in vivo or expand CAR⁺ CD8 T cell counts. FIG. 16A: Endpointperipheral blood CD4 T cell counts were collected 22 days post HIVinfection, as shown in FIG. 12B, but now includes data from mice thatreceived CCR5 ZFN modified CD8 T cells as well. (B) Endpoint peripheralblood CAR⁺ CD8 T cell counts were enumerated 22 days post infection inall cohorts. Significance was detected using a Mann Whitney Test (pvalues: ns>0.05, *<0.05, **<0.01, ***<0.0001).

FIGS. 17A-17D are series of tables and graphs summarizing theimprovements made in this invention to the original clinical trialvector. FIG. 17A: Table and schematic depicting the complete list ofmodifications explored to improve the original clinical trial,MMLV-based construct. FIG. 17B: Using the experimental design summarizedin FIG. 2C, primary human CD8 T cells were activated with αCD3/αCD28coated beads and were either left non-transduced (NTD), transduced withthe original MMLV-based CD4 based CAR driven by the PGK promoter(clinical trial CAR), or transduced the optimized EF1α-CD8α TM CAR,placed in a HIV-based lentiviral vector. Transduction efficiencies werenormalized to 60% prior to co-culture. After 7 days of co-culture withHIV Bal-infected CD4 T cells, the expression of surface CD4 andintracellular Gag p24 was measured by flow cytometry, gating on CD8negative T cells. FIG. 17C: Shows gating on the CD8 positive cells. FIG.17D: Summary data for a single experiment performed in triplicate,gating on the CD8 negative cells. Error bars indicate standard error ofthe mean (SEM). This data is representative of three independentexperiments.

FIG. 18 is a list of annotated amino acid sequences for some of theoptimized HIV CD4 CARs and HIV antibody based CARs (SEQ ID NOs: 44-61)of this invention.

FIG. 19 is a list of annotated nucleic acid sequences for some of theoptimized HIV CD4 CARs and HIV antibody based CARs (SEQ ID NOs: 63-79)of this invention.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.10% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

As used herein, the term “adaptor molecule” refers to a polypeptide witha sequence that permits interaction with two or more molecules, and incertain embodiments, promotes activation or inactivation of a cytotoxiccell.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies (scFv) andhumanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. α and β light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as beingforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addison's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),epididymitis, glomerulonephritis, Graves' disease, Guillain-Barresyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, pemphigusvulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis,vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis,among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “broadly neutralizing antibody (bnAb)” refers to an antibodythat defends a cell from multiple strains of a particular virus byneutralizing its effect. In some embodiments, broadly neutralizing HIV-1Antibodies (bnAbs) are neutralizing antibody which neutralize multipleHIV-1 viral strain.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

By the term “CD4” as used herein is meant any amino acid sequencespecifying CD4 from any source, including an amino acid sequence of CD4that has been generated through codon optimization of the nucleic acidsequence encoding CD4. Codon optimization may be accomplished using anyavailable technology and algorithms designed to optimize codons in anamino acid sequence.

The term “chimeric antigen receptor” or “CAR,” as used herein, refers toan artificial T cell receptor that is engineered to be expressed on animmune effector cell and specifically bind an antigen. CARs may be usedas a therapy with adoptive cell transfer. T cells are removed from apatient and modified so that they express the receptors specific to aparticular form of antigen. In some embodiments, the CARs have beenexpressed with specificity to a tumor associated antigen, for example.CARs may also comprise an intracellular activation domain, atransmembrane domain and an extracellular domain comprising a tumorassociated antigen binding region. In some aspects, CARs comprisefusions of single-chain variable fragments (scFv) derived monoclonalantibodies, fused to transmembrane and intracellular domain. Thespecificity of CAR designs may be derived from ligands of receptors(e.g., peptides). In some embodiments, a CAR can target HIV infectedcells by redirecting the specificity of a T cell expressing the CARspecific for HIV associated antigens.

The term “chimeric intracellular signaling molecule” refers torecombinant receptor comprising one or more intracellular domains of oneor more co-stimulatory molecules. The chimeric intracellular signalingmolecule substantially lacks an extracellular domain. In someembodiments, the chimeric intracellular signaling molecule comprisesadditional domains, such as a transmembrane domain, a detectable tag,and a spacer domain.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for the ability to bind antigensusing the functional assays described herein.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86,common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIa,DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically bindswith CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2Rgamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,CD49f, ITGAD, CD1 d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b,ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46,NKG2D, other co-stimulatory molecules described herein, any derivative,variant, or fragment thereof, any synthetic sequence of a co-stimulatorymolecule that has the same functional capability, and any combinationthereof.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

The term “cytotoxic” or “cytotoxicity” refers to killing or damagingcells. In one embodiment, cytotoxicity of the modified cells isimproved, e.g. increased cytolytic activity of T cells.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited to,anti-tumor activity as determined by any means suitable in the art.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of T cells. In one embodiment, the T cellsthat are expanded ex vivo increase in number relative to the numberoriginally present in the culture. In another embodiment, the T cellsthat are expanded ex vivo increase in number relative to other celltypes in the culture. The term “ex vivo,” as used herein, refers tocells that have been removed from a living organism, (e.g., a human) andpropagated outside the organism (e.g., in a culture dish, test tube, orbioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

By the terms “Human Immunodeficiency Virus” or HIV” as used herein ismeant any HIV strain or variant that is known in the art or that isheretofore unknown, including without limitation, HIV-1 and HIV-2.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous. As applied to thenucleic acid or protein, “homologous” as used herein refers to asequence that has about 50% sequence identity. More preferably, thehomologous sequence has about 75% sequence identity, even morepreferably, has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anArginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

The guide nucleic acid sequence may be complementary to one strand(nucleotide sequence) of a double stranded DNA target site. Thepercentage of complementation between the guide nucleic acid sequenceand the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%. The guide nucleic acid sequence can be at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35 or more nucleotides in length. In someembodiments, the guide nucleic acid sequence comprises a contiguousstretch of 10 to 40 nucleotides. The variable targeting domain can becomposed of a DNA sequence, a RNA sequence, a modified DNA sequence, amodified RNA sequence (see for example modifications described herein),or any combination thereof.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

“KIR” means killer cell immunoglobulin-like receptor, KIRs have beencharacterized in humans and non-human primates, and are polymorphic type1 trans-membrane molecules present on certain subsets of lymphocytes,including NK cells and some T cells. KIRs regulate the killing functionof NK cells by interacting with determinants in the alpha 1 and 2domains of the MHC class I molecules. This interaction allows them todetect virus infected cells or tumor cells.

Most KIRs are inhibitory, meaning that their recognition of MHCsuppresses the cytotoxic activity of the NK cell that expresses them.Only a limited number of KIRs have the ability to activate cells. TheKIR gene family has at least 15 gene loci (KIR2DL1, KIR2DL2/L3, KIR2DL4,KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5,KIR3DL1/S1, KIR3DL2, KIR3DL3) and two pseudogenes (KIR2DP1 and KIR3DP1)encoded within a 100-200 Kb region of the Leukocyte Receptor Complex(LRC) located on chromosome 19 (19q13.4). The LRC constitutes a large, 1Mb, and dense cluster of rapidly evolving immune genes which containsgenes encoding other cell surface molecules with distinctive Ig-likeextracellular domains. In addition, the extended LRC contains genesencoding the transmembrane adaptor molecules DAP10 and DAP12.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of a tumorantigen is intended to indicate an abnormal level of expression of atumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

The term “resistance to immunosuppression” refers to lack of suppressionor reduced suppression of an immune system activity or activation.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the plasma membrane of a cell.

“Single chain antibodies” refer to antibodies formed by recombinant DNAtechniques in which immunoglobulin heavy and light chain fragments arelinked to the Fv region via an engineered span of amino acids. Variousmethods of generating single chain antibodies are known, including thosedescribed in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward etal. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

As used herein, the term “substantially lacks an extracellular domain”refers to a molecule that is essentially free of a domain that extrudesextracellularly. In one embodiment, the chimeric intracellular signalingmolecule lacks any function performed by an extracellular domain, suchas antigen binding. In another embodiment, the chimeric intracellularsignaling molecule includes a transmembrane domain but lacks afunctional extracellular domain.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

A “target site” or “target sequence” refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (a) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the ICR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “tumor” as used herein, refers to an abnormal growth of tissuethat may be benign, pre-cancerous, malignant, or metastatic.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention includes compositions and methods for thetreatment of HIV infection using a CD4 membrane-bound chimeric receptoror HIV specific scFV chimeric antigen receptors (CARs). According to theinvention, T cells are modified for adoptive T cell therapy byexpressing a fragment of CD4 or scFVs capable of specificallyrecognizing and binding HIV-infected cells. The modified T cells of thisinvention are specific for HIV infected cells and have improvedcytotoxicity and efficacy against HIV infection.

HIV Specific CD4 Membrane-Bound Chimeric Receptor

The present invention includes a membrane-bound chimeric receptorcomprising a CD4 domain, particularly a CD4 extracellular domain thatspecifically binds to HIV virions or HIV infected cells. In certainembodiments, the CD4 membrane-bound chimeric receptor of the inventioncomprises particular structural features such as particular amino acidsequences or peptides as disclosed herein. The invention also includesmethods of making such a receptor. The membrane-bound chimeric receptorof the invention can be incorporated into a pharmaceutical compositionfor use in treating a subject, for example for use as an immunotherapy.Accordingly, the present invention provides compositions and methods fortreating HIV infection or HIV related diseases.

In one aspect the invention includes an isolated nucleic acid sequenceencoding a membrane-bound chimeric receptor comprising a CD4extracellular domain, a transmembrane domain, and a signaling domain,wherein the CD4 extracellular domain is capable of recognizing andbinding a HIV infected cell. In another aspect, the invention includesan isolated amino acid sequence encoding a membrane-bound chimericreceptor comprising a CD4 extracellular domain, a transmembrane domain,and a signaling domain, wherein the CD4 extracellular domain is capableof recognizing and binding a HIV infected cell.

In one embodiment, the CD4 extracellular domain comprises SEQ ID NO: 3,46 or 64, the transmembrane domain comprises a CD8alpha hinge (SEQ IDNO: 4, 47 or 65) and a transmembrane domain comprising at least onedomain selected from the group consisting of CD8alpha transmembranedomain (SEQ ID NO: 5, 48 or 66) and CD28 transmembrane domain (SEQ IDNO: 8, 49 or 67). In another embodiment, the signaling domain comprisesa CD3zeta signaling domain (SEQ ID NO: 6, 51 or 68). In yet anotherembodiment, the costimulatory signaling region comprises anintracellular domain of a costimulatory molecule selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83, and anycombination thereof. In yet another embodiment, the costimulatorysignaling region CD28 (SEQ ID NO: 9, 49 or 67) is present in theconstruct. In a further embodiment, CD4 extracellular domainspecifically binds to the HIV envelope (Env) glycoprotein.

In one aspect, the invention includes a vector comprising a eukaryoticelongation factor (EF1 alpha) promoter, a CD4 extracellular domain, aCD8alpha hinge, a CD8alpha transmembrane domain and CD3zeta signalingdomain (SEQ ID NO: 1). In another aspect, the invention includes avector comprising an EF1α promoter, a CD4 extracellular domain, aCD8alpha hinge, a CD28 transmembrane domain, a CD28 costimulatory domainand CD3zeta signaling domain (SEQ ID NO: 7). In yet another aspect, theinvention includes a vector comprising a membrane-bound chimericreceptor comprising a CD4 extracellular domain, a transmembrane domain,and a signaling domain, wherein the CD4 extracellular domain is capableof recognizing and binding a HIV infected cell, wherein the vectorcomprises at least one amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44, 45, 62 and 63.

In one embodiment, the membrane-bound chimeric receptor is encoded bythe nucleic acid sequence of SEQ ID NO: 30, 32 or 64. In anotherembodiment, the membrane-bound chimeric receptor has an amino acidsequence of SEQ ID NO: 31, 33 or 46.

CD4 Extracellular Domain

CD4 is a member of the immunoglobulin superfamily and includes fourextracellular immunoglobulin domains (D1 to D4). D1 and D3 are similarto immunoglobulin variable domains and D2 and D4 are similar toimmunoglobulin constant domains. D1 includes the region of CD4 thatinteracts with beta2-microglobulin of the major histocompatibilitycomplex class II molecules.

In one embodiment, the membrane-bound chimeric receptor comprises anextracellular domain of CD4 or a fragment thereof. In anotherembodiment, the membrane-bound chimeric receptor comprises at least oneimmunoglobulin domain of CD4. In another embodiment, the CD4extracellular domain comprises SEQ ID NO: 3 or 64.

The CD4 extracellular domain described herein, such as at least oneimmunoglobulin domain of CD4 or the CD4 extracellular domain comprisingSEQ ID NO: 3 or 64, can be combined with any of the transmembranedomains described herein, any of the signaling domains described herein,or any of the other domains described herein that may be included in themembrane-bound chimeric receptor.

Transmembrane Domain

In one embodiment, the transmembrane domain is associated with one ofthe domains in the CD4 chimeric antigen receptor construct. In someinstances, the transmembrane domain can be selected or modified by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use may be derived from (i.e. comprise at least thetransmembrane region(s) of) the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some instances, avariety of human hinges can be employed as well including the human Ig(immunoglobulin) hinge. In one embodiment, the transmembrane domaincomprises a CD8alpha hinge and transmembrane domain.

In one embodiment, the transmembrane domain comprises a CD8alphatransmembrane domain (SEQ ID NO: 5 or 66). In another embodiment, thetransmembrane domain comprises a CD28 transmembrane domain (SEQ ID NO: 8or 67). In yet another embodiment, the transmembrane domain comprises ahinge domain, such as a CD8alpha hinge (SEQ ID NO: 4 or 65). Thetransmembrane domain may be combined with any hinge domain and/or maycomprise one or more transmembrane domains described herein.

The transmembrane domains described herein, such as at least atransmembrane region(s) of the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, or CD154, can be combined with anyof the CD4 extracellular domain described herein, any of the signalingdomains described herein, or any of the other domains described hereinthat may be included in the membrane-bound chimeric receptor.

In another embodiment, the transmembrane domain may be synthetic, inwhich case it comprises predominantly hydrophobic residues such asleucine and valine. Preferably a triplet of phenylalanine, tryptophanand valine is present at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain. Aglycine-serine doublet provides a particularly suitable linker.

Signaling Domain

The signaling domain or intracellular signaling domain is responsiblefor activation of at least one of the normal effector functions of theimmune cell in which the CD4 chimeric antigen receptor construct isexpressed. The term “effector function” refers to a specialized functionof a cell. Effector function of a T cell, for example, may be cytolyticactivity or helper activity including the secretion of cytokines. Thusthe term “intracellular signaling domain” or “signaling domain” refersto the portion of a protein which transduces the effector functionsignal and directs the cell to perform a specialized function. Whileusually the entire signaling domain can be employed, in many cases it isnot necessary to use the entire molecule. To the extent that a truncatedportion of the signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term signaling domain is thus meant to include anytruncated portion of the signaling domain sufficient to transduce theeffector function signal.

Examples of signaling domains for use in this invention include thecytoplasmic sequences of the T cell receptor (TCR) and co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement, as well as any derivative or variant of thesesequences and any synthetic sequence that has the same functionalcapability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the immunoreceptor of the invention comprises acytoplasmic signaling sequence derived from CD3-zeta.

In a preferred embodiment, the signaling domain of the CD4 chimericantigen receptor construct can be designed to comprise the CD3-zetasignaling domain by itself or combined with any other desired signalingdomain(s) useful in the context of the immunoreceptor. For example, thesignaling domain of the immunoreceptor can comprise a CD3 zeta chainportion and a costimulatory signaling region. The costimulatorysignaling region refers to a portion of the immunoreceptor comprisingthe intracellular domain of a costimulatory molecule. A costimulatorymolecule is a cell surface molecule other than an antigen receptor orits ligands that is required for an efficient response of lymphocytes toan antigen. Examples of such molecules include CD27, CD28, 4-1BB(CD137), DAP12, OX9, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with CD28 as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

In one embodiment, the signaling domain comprises a CD3zeta signalingdomain (SEQ ID NO: 6, 51 or 68). In another embodiment, the signalingdomain comprises a CD4 CD28 zeta (SEQ ID NO:7 or 62). In anotherembodiment, the signaling domain comprises a CD28 costimulatory domain(SEQ ID NO:9 or 67). In yet another embodiment, the signaling domaincomprises a CD4 4-1BB costimulatory domain (SEQ ID NO: 63). In yetanother embodiment, the signaling domain comprises a 4-1BB (CD137)costimulatory domain (SEQ ID NO:69). The signaling domain may becomprise one or more signaling domains described herein.

The signaling domains described herein, such as cytoplasmic signalingsequences of the alpha, beta or zeta chain of the TCR zeta, FcR gamma,FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, orCD66d or a costimulatory signaling region from CD27, CD28, 4-1BB(CD137), DAP12, OX9, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ora ligand that specifically binds with CD83, can be combined with any ofthe CD4 extracellular domain described herein, any of the transmembranedomains described herein, or any of the other domains described hereinthat may be included in the membrane-bound chimeric receptor.

Chimeric Antigen Receptor (CAR)

Without wishing to be bound by theory, it is understood that use of aCAR that targets HIV, wherein the CAR comprises an anti-HIV scFv will bebeneficial for treatment of subjects that have reservoir populations ofHIV-infected cells. The invention therefore includes such a CAR for suchuse in one aspect of the invention.

Thus, in one aspect of the invention, a T cell is generated byexpressing a CAR therein. Thus, the present invention encompasses a CARand a nucleic acid construct encoding a CAR, wherein the CAR includes anantigen binding domain (e.g., an scFv encoding an anti-HIV antibody), atransmembrane domain and an intracellular domain.

In one aspect, the invention includes a modified cell comprising achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain and an intracellular domain of aco-stimulatory molecule, and wherein the cell is a T cell that possessestargeted effector activity. In another aspect, the invention includes amodified cell comprising a nucleic acid sequence encoding a chimericantigen receptor (CAR), wherein nucleic acid sequence comprises anucleic acid sequence encoding an antigen binding domain, a nucleic acidsequence encoding a transmembrane domain and a nucleic acid sequenceencoding an intracellular domain of a co-stimulatory molecule, andwherein the cell is a T cell that expresses the CAR and possessestargeted effector activity (e.g. targeted cellular cytotoxicity andantigen presentation). In one embodiment, the targeted effector activityis directed against an antigen on a target cell that specifically bindsthe antigen binding domain of the CAR. In one aspect, the target antigenis a HIV specific antigen and the CAR comprises a HIV specific bindingdomain comprising an anti-HIV antibody or fragment thereof.

In one embodiment, the CAR is encoded by a nucleic acid sequenceselected from SEQ ID NO:10, 14, 18, 22, 26, 34, 70-73 and 74. In anotherembodiment, the CAR has an amino acid sequence of SEQ ID NO:11, 15, 19,23, 27, 35, 52-55, or 56.

Antigen Binding Domain

In one embodiment, the antigen binding domain of the invention comprisesa HIV specific binding domain that binds to HIV virions, such as HIVenvelope glycoprotein (Env) (e.g. gp120), and/or HIV infected cell.Examples of other cell surface markers that may act as an antigeninclude those associated with viral, bacterial and parasitic infections,autoimmune disease, and cancer cells. In another embodiment, the antigenbinding domain of the invention comprises an antibody or fragmentthereof that binds to a HIV protein, such as a scFv antibody.Preferably, the antigen binding domain is scFv antibody that binds to aHIV protein, for example, HIV Env. Specific scFvs that are useful arethose shown in FIG. 4A, FIG. 8, FIG. 18 and FIG. 19 (SEQ ID NO:11, 12,16, 17, 20, 21, 24, 25, 28, 29, 57-61, 75-78, or 79).

The choice of antigen binding domain depends upon the type and number ofantigens that are present on the surface of a target cell. For example,the antigen binding domain may be chosen to recognize an antigen thatacts as a cell surface marker on a target cell associated with aparticular disease state.

The antigen binding domain can include any domain that binds to theantigen and may include, but is not limited to, a monoclonal antibody, apolyclonal antibody, a synthetic antibody, a human antibody, a humanizedantibody, a non-human antibody, and any fragment thereof. Thus, in oneembodiment, the antigen binding domain portion comprises a mammalianantibody or a fragment thereof. In another embodiment, the antigenbinding domain of the CAR is selected from the group consisting of ananti-HIV antibody and a fragment thereof.

In some instances, it is beneficial for the antigen binding domain to bederived from the same species in which the CAR will ultimately be usedin. For example, for use in humans, it may be beneficial for the antigenbinding domain of the CAR to comprise a human antibody, as describedelsewhere herein, or a fragment thereof.

It is also beneficial that the antigen binding domain is operably linkedto another domain of the CAR, such as the transmembrane domain or theintracellular domain, both described elsewhere herein, for expression inthe cell. In one embodiment, a nucleic acid encoding the antigen bindingdomain is operably linked to a nucleic acid encoding a transmembranedomain and a nucleic acid encoding an intracellular domain.

The antigen binding domains described herein, such as the antibody orfragment thereof that binds to a HIV protein, can be combined with anyof the transmembrane domains described herein, any of the intracellulardomains or cytoplasmic domains described herein, or any of the otherdomains described herein that may be included in the CAR.

Transmembrane Domain

With respect to the transmembrane domain, the CAR (or the membrane-boundchimeric receptor construct) can be designed to comprise a transmembranedomain that connects the antigen binding domain of the CAR to theintracellular domain. In one embodiment, the transmembrane domain isnaturally associated with one or more of the domains in the CAR. In someinstances, the transmembrane domain can be selected or modified by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-likereceptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.In some instances, a variety of hinges can be employed as well includingthe Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain comprises a CD8alphatransmembrane domain (SEQ ID NOs: 5, 48, 66). In another embodiment, thetransmembrane domain comprises a CD28 transmembrane domain (SEQ ID NOs:8, 49 and 67). In yet another embodiment, the transmembrane domaincomprises a hinge domain, such as a CD8alpha hinge (SEQ ID NO: 4, 47 and65). The transmembrane domain may be combined with any hinge domainand/or may comprise one or more transmembrane domains described herein.

The transmembrane domains described herein, such as a transmembraneregion of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, can be combined with any of theantigen binding domains described herein, any of the intracellulardomains or cytoplasmic domains described herein, or any of the otherdomains described herein that may be included in the CAR.

In one embodiment, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.

Intracellular Domain

The intracellular domain or otherwise the cytoplasmic domain of the CAR(or the membrane-bound chimeric receptor construct) includes a similaror the same intracellular domain as the chimeric intracellular signalingmolecule described elsewhere herein, and is responsible for activationof the cell in which the CAR is expressed.

In one embodiment, the intracellular domain of the CAR includes a domainresponsible for signal activation and/or transduction.

Examples of an intracellular domain for use in the invention include,but are not limited to, the cytoplasmic portion of a surface receptor,co-stimulatory molecule, and any molecule that acts in concert toinitiate signal transduction in the T cell, as well as any derivative orvariant of these elements and any synthetic sequence that has the samefunctional capability.

Examples of the intracellular domain include a fragment or domain fromone or more molecules or receptors including, but are not limited to,TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40,CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory moleculesdescribed herein, any derivative, variant, or fragment thereof, anysynthetic sequence of a co-stimulatory molecule that has the samefunctional capability, and any combination thereof.

In one embodiment, the intracellular domain of the CAR includes anyportion of one or more co-stimulatory molecules, such as at least onesignaling domain from CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, anyderivative or variant thereof, any synthetic sequence thereof that hasthe same functional capability, and any combination thereof.

In one embodiment, the intracellular domain comprises a CD3zetasignaling domain (SEQ ID NO: 6, 51 or 68). In another embodiment, theintracellular domain comprises a CD4 CD28 zeta, (SEQ ID NO:7, or 62). Inanother embodiment, the intracellular domain comprises a CD28costimulatory domain (SEQ ID NO: 9 or 67). In yet another embodiment,the signaling domain comprises a CD4 4-1BB costimulatory domain (SEQ IDNO:63). In still another embodiment, the signaling domain comprises a4-1BB (CD137) costimulatory domain (SEQ ID NO:69). In anotherembodiment, the intracellular domain comprises a DAP12 domain. In yetanother embodiment, the intracellular domain comprises a KIR domain. Theintracellular domain may comprise one or more intracellular domainsdescribed herein described herein.

The intracellular domains described herein, such as a fragment or domainfrom TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40,CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or other co-stimulatorymolecules, can be combined with any of the antigen binding domainsdescribed herein, any of the transmembrane domains described herein, orany of the other domains described herein that may be included in theCAR.

Between the antigen binding domain and the transmembrane domain of theCAR, or between the intracellular domain and the transmembrane domain ofthe CAR, a spacer domain may be incorporated. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to, either the antigen binding domainor, the intracellular domain in the polypeptide chain. In oneembodiment, the spacer domain may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. In another embodiment, a short oligo- or polypeptide linker,preferably between 2 and 10 amino acids in length may form the linkagebetween the transmembrane domain and the intracellular domain of theCAR. An example of a linker includes a glycine-serine doublet.

Human Antibodies

It may be preferable to use human antibodies or fragments thereof whenusing the antigen binding domains of a CAR. Completely human antibodiesare particularly desirable for therapeutic treatment of human subjects.Human antibodies can be made by a variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences, including improvements to thesetechniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Antibodies directed against the target ofchoice can be obtained from the immunized, transgenic mice usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies, including, but not limited to, IgG1(gamma 1) and IgG3. For an overview of this technology for producinghuman antibodies, see, Lonberg and Huszar (Int. Rev. Immunol., 13:65-93(1995)). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893,WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598,each of which is incorporated by reference herein in their entirety. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above. For a specific discussion of transfer of a humangerm-line immunoglobulin gene array in germ-line mutant mice that willresult in the production of human antibodies upon antigen challenge see,e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309(1996)). Phage display technology (McCafferty et al., Nature,348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of unimmunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J.,12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905,each of which is incorporated herein by reference in its entirety.

Human antibodies may also be generated by in vitro activated B cells(see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies mayalso be generated in vitro using hybridoma techniques such as, but notlimited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

Humanized Antibodies

Alternatively, in some embodiments, a non-human antibody can behumanized, where specific sequences or regions of the antibody aremodified to increase similarity to an antibody naturally produced in ahuman. For instance, in the present invention, the antibody or fragmentthereof may comprise a non-human mammalian scFv. In one embodiment, theantigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some framework(FR) residues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety).

Antibodies can be humanized with retention of high affinity for thetarget antigen and other favorable biological properties. According toone aspect of the invention, humanized antibodies are prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind the target antigen.In this way, FR residues can be selected and combined from the recipientand import sequences so that the desired antibody characteristic, suchas increased affinity for the target antigen, is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding.

A humanized antibody retains a similar antigenic specificity as theoriginal antibody. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody to the targetantigen may be increased using methods of “directed evolution,” asdescribed by Wu et al., J. Mol. Biol., 294:151 (1999), the contents ofwhich are incorporated herein by reference herein in their entirety.

Vectors

A vector may be used to introduce the chimeric intracellular signalingmolecule or the CAR into a T cell as described elsewhere herein. In oneaspect, the invention includes a vector comprising a nucleic acidsequence encoding a chimeric intracellular signaling. In another aspect,the invention includes a vector comprising a nucleic acid sequenceencoding a CAR. In one embodiment, the vector comprises a plasmidvector, viral vector, retrotransposon (e.g. piggyback, sleeping beauty),site directed insertion vector (e.g. CRISPR, zn finger nucleases,TALEN), or suicide expression vector, or other known vector in the art.

All constructs mentioned above are capable of use with 3rd generationlentiviral vector plasmids, other viral vectors, or RNA approved for usein human cells. In one embodiment, the vector is a viral vector, such asa lentiviral vector. In another embodiment, the vector is a RNA vector.

The production of any of the molecules described herein can be verifiedby sequencing. Expression of the full length proteins may be verifiedusing immunoblot, immunohistochemistry, flow cytometry or othertechnology well known and available in the art.

The present invention also provides a vector in which DNA of the presentinvention is inserted. Vectors, including those derived fromretroviruses such as lentivirus, are suitable tools to achieve long-termgene transfer since they allow long-term, stable integration of atransgene and its propagation in daughter cells. Lentiviral vectors havethe added advantage over vectors derived from onco-retroviruses, such asmurine leukemia viruses, in that they can transduce non-proliferatingcells, such as hepatocytes. They also have the added advantage ofresulting in low immunogenicity in the subject into which they areintroduced.

The expression of natural or synthetic nucleic acids is typicallyachieved by operably linking a nucleic acid or portions thereof to apromoter, and incorporating the construct into an expression vector. Thevector is one generally capable of replication in a mammalian cell,and/or also capable of integration into the cellular genome of themammal. Typical vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The nucleic acid can be cloned into any number of different types ofvectors. For example, the nucleic acid can be cloned into a vectorincluding, but not limited to a plasmid, a phagemid, a phage derivative,an animal virus, and a cosmid. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors.

The expression vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the EF-1 alpha promoter, as well as human gene promoterssuch as, but not limited to, the actin promoter, the myosin promoter,the hemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess expression of a polypeptide or portions thereof, theexpression vector to be introduced into a cell can also contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes, such as neo and thelike.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assessed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Introduction of Nucleic Acids

Methods of introducing and expressing genes, such as the chimericintracellular signaling molecule or the CAR, into a cell are known inthe art. In the context of an expression vector, the vector can bereadily introduced into a host cell, e.g., mammalian, bacterial, yeast,or insect cell by any method in the art. For example, the expressionvector can be transferred into a host cell by physical, chemical, orbiological means.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). Nucleic acids can be introduced into target cells usingcommercially available methods which include electroporation (AmaxaNucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX)(Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad,Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). Nucleic acidscan also be introduced into cells using cationic liposome mediatedtransfection using lipofection, using polymer encapsulation, usingpeptide mediated transfection, or using biolistic particle deliverysystems such as “gene guns” (see, for example, Nishikawa, et al. HumGene Ther., 12(8):861-70 (2001).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. RNA vectors includevectors having a RNA promoter and/other relevant domains for productionof a RNA transcript. Viral vectors, and especially retroviral vectors,have become the most widely used method for inserting genes intomammalian, e.g., human cells. Other viral vectors may be derived fromlentivirus, poxviruses, herpes simplex virus, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the molecules describedherein, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, “molecular biological” assays well known to those of skillin the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

In one embodiment, one or more of the nucleic acid sequences describedelsewhere herein are introduced by a method selected from the groupconsisting of transducing the population of cells, transfecting thepopulation of cells, and electroporating the population of cells. In oneembodiment, a population of cells comprises one or more of the nucleicacid sequences described herein.

In one embodiment, the nucleic acids introduced into the cell are RNA.In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA is produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired template for in vitrotranscription is a chimeric intracellular signaling molecule.

PCR can be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary”, as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary, or one or more basesare non-complementary, or mismatched. Substantially complementarysequences are able to anneal or hybridize with the intended DNA targetunder annealing conditions used for PCR. The primers can be designed tobe substantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100 T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

Some in vitro-transcribed RNA (IVT-RNA) vectors are known in theliterature which are utilized in a standardized manner as template forin vitro transcription and which have been genetically modified in sucha way that stabilized RNA transcripts are produced. Currently protocolsused in the art are based on a plasmid vector with the followingstructure: a 5′ RNA polymerase promoter enabling RNA transcription,followed by a gene of interest which is flanked either 3′ and/or 5′ byuntranslated regions (UTR), and a 3′ polyadenyl cassette containing50-70 A nucleotides. Prior to in vitro transcription, the circularplasmid is linearized downstream of the polyadenyl cassette by type IIrestriction enzymes (recognition sequence corresponds to cleavage site).The polyadenyl cassette thus corresponds to the later poly(A) sequencein the transcript. As a result of this procedure, some nucleotidesremain as part of the enzyme cleavage site after linearization andextend or mask the poly(A) sequence at the 3′ end. It is not clear,whether this nonphysiological overhang affects the amount of proteinproduced intracellularly from such a construct.

In one aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

Alternatively, in another aspect, the invention includes a method forgenerating a modified T cell comprising electroporating a population ofT cells with a nucleic acid sequence encoding a chimeric intracellularsignaling molecule, wherein the nucleic acid sequence comprises anucleic acid sequence of an intracellular domain of a co-stimulatorymolecule and substantially lacks an extracellular domain. In oneembodiment, the nucleic acid sequence encoding a chimeric intracellularsignaling molecule is electroporated into a cell. In yet anotherembodiment, a nucleic acid sequence encoding a CAR or a membrane-boundchimeric receptor is further electroporated into the cell.

Sources of T Cells

The modified T cells may be generated from any source of T cells. In oneembodiment, a source of T cells is obtained from a subject. Non-limitingexamples of subjects include humans, dogs, cats, mice, rats, andtransgenic species thereof. Preferably, the subject is a human. T cellscan be obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, spleen tissue,umbilical cord, and tumors. In certain embodiments, any number of T celllines available in the art, may be used. In certain embodiments, T cellscan be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficollseparation. In one embodiment, cells from the circulating blood of anindividual are obtained by apheresis or leukapheresis. The apheresisproduct typically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. The cells collected by apheresis may be washed toremove the plasma fraction and to place the cells in an appropriatebuffer or media, such as phosphate buffered saline (PBS) or washsolution lacks calcium and may lack magnesium or may lack many if notall divalent cations, for subsequent processing steps. After washing,the cells may be resuspended in a variety of biocompatible buffers, suchas, for example, Ca-free, Mg-free PBS. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from umbilical cord. In any event, a specific subpopulationof T cells can be further isolated by positive or negative selectiontechniques.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19 and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4+ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11 b,CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80° C. at arate of 1° per minute and stored in the vapor phase of a liquid nitrogenstorage tank. Other methods of controlled freezing may be used as wellas uncontrolled freezing immediately at −20° C. or in liquid nitrogen.

In one embodiment, a population of cells comprise the T cells of thepresent invention. Examples of a population of cells include, but arenot limited to, peripheral blood mononuclear cells, cord blood cells, apurified population of T cells, and a T cell line. In anotherembodiment, peripheral blood mononuclear cells comprise the populationof T cells. In yet another embodiment, purified T cells comprise thepopulation of T cells.

Expansion of T Cells

T cells generated by any method described herein may be expanded exvivo. In one embodiment, T cells or a population of cells comprising Tcells are cultured for expansion. Generally, T cells are expanded bycontact with a surface having attached thereto an agent that stimulatesa CD3/TCR complex associated signal and a ligand that stimulates aco-stimulatory molecule on the surface of the T cells.

Methods for expanding T cells are described herein. For example, the Tcells can be expanded by about 10 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold,400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold,2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000fold, or greater, and any and all whole or partial integerstherebetween. In one embodiment, the T cells expand in the range ofabout 20 fold to about 50 fold.

The T cells can be incubated in cell medium in a culture apparatus for aperiod of time or until the cells reach confluency or high cell densityfor optimal passage before passing the cells to another cultureapparatus. The culturing apparatus can be of any culture apparatuscommonly used for culturing cells in vitro. Preferably, the level ofconfluence is 70% or greater before passing the cells to another cultureapparatus. More preferably, the level of confluence is 90% or greater. Aperiod of time can be any time suitable for the culture of cells invitro. The T cell medium may be replaced during the culture of the Tcells at any time. Preferably, the T cell medium is replaced about every2 to 3 days. The T cells are then harvested from the culture apparatuswhereupon the T cells can be used immediately or cryopreserved to bestored for use at a later time. In one embodiment, the inventionincludes cryopreserving the expanded T cells. The cryopreserved T cellsare thawed prior to introducing one or more of the molecules describedelsewhere herein into the T cells.

The culturing step as described herein (contact with agents as describedherein) can be very short, for example less than 24 hours such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,or 23 hours. The culturing step as described further herein (contactwith agents as described herein) can be longer, for example 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

In one embodiment, the T cells may be cultured for several hours (about3 hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-α. or any other additives for the growthof cells known to the skilled artisan. Other additives for the growth ofcells include, but are not limited to, surfactant, plasmanate, andreducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Mediacan include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, andX-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

The T cell culturing medium may include an agent that can co-stimulatethe T cells. For example, an agent that can stimulate CD3 is an antibodyto CD3, and an agent that can stimulate CD28 is an antibody to CD28.This is because, as demonstrated by the data disclosed herein, a cellisolated by the methods disclosed herein can be expanded approximately10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold,90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold,5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold,100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In oneembodiment, the T cells expand in the range of about 20 fold to about 50fold, or more by culturing the electroporated population.

Therapy

In one aspect, the invention includes a method of treating a disease orcondition associated with HIV infection in a subject comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising the modified T cell describedherein.

The modified T cells as described herein can be administered to ananimal, preferably a mammal, even more preferably a human, to treat HIVinfection. In addition, the modified T cells of the present inventioncan be used for the treatment of any condition in which a diminished orotherwise inhibited immune response, especially a cell-mediated immuneresponse, is desirable to treat or alleviate the disease.

The administration of the modified T cells of the invention may becarried out in any convenient manner known to those of skill in the art.The modified T cells of the present invention may be administered to asubject by aerosol inhalation, injection, ingestion, transfusion,implantation or transplantation. The compositions described herein maybe administered to a patient transarterially, subcutaneously,intradermally, intratumorally, intranodally, intramedullary,intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.In other instances, the modified T cells of the invention are injecteddirectly into a site of inflammation in the subject, a local diseasesite in the subject, a lymph node, an organ, a tumor, and the like.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise themodified T cells as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arepreferably formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-immune responseeffective amount”, “an immune response-inhibiting effective amount”, or“therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, immune response, and condition of the patient (subject).It can generally be stated that a pharmaceutical composition comprisingthe T cells described herein may be administered at a dosage of 10⁴ to10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. T cell compositionsmay also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer the modified Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), metabolically enhance T cells therefrom accordingto the present invention, and reinfuse the patient with these modified Tcells. This process can be carried out multiple times every few weeks.In certain embodiments, T cells can be obtained from blood draws fromabout 10 ml to about 400 ml. In certain embodiments, modified T cellsare obtained from blood draws of about 20 ml, 30 ml, 40 ml, 50 ml, 60ml, 70 ml, 80 ml, 90 ml, or 100 ml. Not to be bound by theory, usingthis multiple blood draw/multiple reinfusion protocol, may select outcertain populations of T cells.

In certain embodiments of the present invention, T cells are modifiedusing the methods described herein, and stimulated, activated orexpanded using the methods described herein or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to any available anti-HIV therapy.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. It should be understood that the method andcompositions that would be useful in the present invention are notlimited to the particular formulations set forth in the examples. Thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the cells, expansion and culture methods, and therapeuticmethods of the invention, and are not intended to limit the scope ofwhat the inventors regard as their invention.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

The materials and methods employed in these experiments are nowdescribed.

Single Chain Variable Fragment (scFv) Based CAR Constructs:

scFvs were synthesized as a single transcript in a 2nd generation CARusing the CD137 and CD3zeta signal transduction domains or as anartificial NK cell receptor by means of KIRS2 together with DAP12. Allconstructs were codon-optimized for expression in humans as onetranscript following a proprietary codon adaptation index (Geneart, LifeTechn.).

The following constructs have been created and tested in this invention:

“PGDM1400” targeting V1/V2 glycans (SEQ ID NOs: 10-13), “PGT128”targeting V3/V4 glycans (SEQ ID NOs: 14-17), “VRC01” targeting CD4binding site (SEQ ID NOs: 18-21 and 34-35), “VRC01 c-mut” (mutation ofnon-canonic disulfide bond between HCDR1 and HCDR3) targeting CD4binding site (SEQ ID NOs: 26-29), “3BNC60” targeting CD4 binding site(SEQ ID NOs: 22-25), and CD4 Dap12-KIRS2 (SEQ ID NOs: 30-31).Vector Construction:

pRT43.2 GFP, the backbone of the original clinical trial vector, wasobtained (Liu and Eiden. Retrovirology 9, 51 (2012)) and a restrictionsite linker was inserted into the PstI and SalI sites, removing the CMVpromoter region. The CD4 zeta sequence expressed under the PGK promoterwas amplified from plasmid pRRL.PGK.F3 with oligos 5′GTATCGATCACGAGACTAGC (SEQ ID NO: 40) and5′TTAAACCGGTGTCTGGCCTTTGAGTGGTGA (SEQ ID NO: 41) and inserted into XhoIand Agel sites in the linker within pRT43.2. pTRPE CD4 zeta was createdby amplifying. The CD4 extracellular domain was amplified by PCR fromthe pRRL.PGK.F3 retroviral backbone containing the original clinicaltrial CD4-CD3 construct with the following primers:

Primer-1 Sense (SEQ ID NO: 36): 5′-TTAATGGGATCCATGAACCGGGGAGTCCCTTT-3′Primer-2 Antisense (SEQ ID NO: 37):5′-AAGGACTTCCGGATGGCTGCACCGGGGTGGACCATG-3′

PCR product was then inserted into the BamHI and BspE1 restriction sitesin the pELNS lentiviral backbone which contained the CD8α extracellularhinge and transmembrane domains and the 4-1BB and CD3 zeta intracellularcostimulatory domains (ICD). pELNS lentiviral vectors containing theCD8α hinge-CD8αTM-CD3ζ or containing the CD8α hinge-CD28TM-CD28-CD3ζ ICDwere obtained and used as template to PCR amplify the hinge-TM-and ICDregion with the following primers:

Primer-3 Sense (SEQ ID NO: 38): 5′-GGGACACTCCGGAACCACGACGCCAGCGCCGCG-3′Primer-4 Antisense (SEQ ID NO: 39): 5′-GGGACACGTCGACTTAGCGAGGGGGCA-3′

This PCR template was then inserted into the BspE1 and Sal1 sites in therecently generated pELNS CD4 4-1BB zeta construct to replace the regioncontaining the CD8α hinge-4-1BB-CD3ζ region. Plasmid was thenelectroporated into and propagated in the Top10 strain of E. coli.

A lentiviral vector that expressed B57 restricted TCR capable ofrecognizing HIV p24Gag epitope KAFSPEVIPMF (SEQ ID NO: 42, pTRPEB57-KF11) was generated by synthesizing the TCRα and TCRβ gene sequence(IDT)). The TCRα and TCRβ gene sequence was separated by the T2A whichallows coordinate expression of both TCR genes as previously described(Varela-Rohena et al., Nature medicine 14, 1390-1395 (2008)). VRC01,3BNC60, PGT128, and PGDM1400 scFv CARs were generated from the publishedparental antibody sequences, with a light-linker-heavy chainconfiguration. The linker sequence is as follows: GGSSRSSSSGGGGSGGGG(SEQ ID NO: 43). Amino acid sequences were codon-optimized (Geneart) andsynthesized as double-stranded DNA fragments (IDT or Geneart), flankedwith suitable restriction sites and then cloned into pTRPE expressionplasmid with the BamHI and BspE1 restriction sites. The PG9 scFv wascloned into the pTRPE expression plasmid with the BamHI and BspE1restriction sites. Some of the sequences of optimized HIV CARs used inthis study are listed in FIG. 18 (SEQ ID NOs: 44-61, amino acids) and inFIG. 19 (SEQ ID NOs: 62-79, nucleic acids).

Construction of VRC01, VRC01c-, and 3BNC60 scFv-Based KIR andIgG4-41BB-Zeta CARs.

VRC01, VRC01c-, 3BNC60 scFvs were cloned into a 3^(rd) generationlentiviral expression vector under control of an EF1α promoter afterdigestion with BamHI and Nhel, so that the scFv is in frame with a 9amino acid GS-linker which is followed by the KIRS2 transmembrane andsignaling domain. DAP-12 is located 5′ of the scFv leader sequence, fromwhich it is separated by a T2A ribosomal skipping site. A similarstrategy was applied to clone the scFv coding sequences into a vectorthat allows expression of the scFvs in frame with an IgG4 hinge,followed by a CD8 transmembrane domain and 41BB co-stimulatory andCD3zeta signaling domain.

Construction of VRC01, 3BNC60, PGT128, PGDA1400 and PG9 scFv-BasedCD3zeta CARs.

VRC01, 3BNC60, PGT128, PGDM1400 and PG9 scFv coding sequences were PCRamplified with primers encoding a 5′ flanking BamHI and a 3′ flankingBspEI site, which allowed digestion and cloning into the aforementionedpELNS CD8α hinge-CD8αTM-CD3ζ between leader sequence and CD8α hinge. Allconstructs were sequence verified.

TABLE 1 Listing of Sequences SEQ ID Number Description 1 CD4 zetaNucleic acid 2 EF1α promoter Nucleic acid 3 CD4 extracellular domainNucleic acid 4 CD8α extracellular hinge Nucleic acid 5 CD8αtransmembrane domain Nucleic acid 6 CD3 zeta Nucleic acid 7 CD4 CD28zeta Nucleic acid 8 CD28 transmembrane domain Nucleic acid 9 CD28costimulatory domain Nucleic acid 10 PGDM1400-KIR Nucleic acid 11PGDM1400-KIR Amino acid 12 PGDM1400 scFv Nucleic acid 13 PGDM1400 scFvAmino acid 14 PGT128-KIR Nucleic acid 15 PGT128-KIR Amino acid 16 PGT128scFv Nucleic acid 17 PGT128 scFv Amino acid 18 VRC01-KIR Nucleic acid 19VRC01-KIR Amino acid 20 VRC01 scFv Nucleic acid 21 VRC01 scFv Amino acid22 3BNC60-KIR Nucleic acid 23 3BNC60-KIR Amino acid 24 3BNC60 scFvNucleic acid 25 3BNC60 scFv Amino acid 26 VRC01c-KIR Nucleic acid 27VRC01c-KIR Amino acid 28 VRC01-c scFv Nucleic acid 29 VRC01-c scFv Aminoacid 30 CD4 Dap12-KIRS2 Nucleic acid 31 CD4 Dap12-KIRS2 Amino acid 32CD4KIR Nucleic acid 33 CD4KIR Amino acid 34 VRC01 IgG4 bbz Nucleic acid35 VRC01 IgG4 bbz Amino acid 44 Optimized CD4-CD28 zeta Amino acid 45Optimized CD4 4-1BB zeta Amino acid 46 Optimized CD4 extracellulardomain Amino acid 47 Optimized CD8α extracellular hinge Amino acid 48Optimized CD8α TM Amino acid 49 Optimized CD28 TM and ICD Amino acid 50Optimized 4-1BB Amino acid 51 Optimized CD3 zeta Amino acid 52 OptimizedPG9 CD3zeta Amino acid 53 Optimized PGT128 CD3zeta Amino acid 54Optimized VRC01 CD3zeta Amino acid 55 Optimized 3BNC60 CD3zeta Aminoacid 56 Optimized PGDM1400 CD3zeta Amino acid 57 Optimized PG9 Aminoacid 58 Optimized PGT128 Amino acid 59 Optimized VRC01 Amino acid 60Optimized 3BNC60 Amino acid 61 Optimized PGDM1400 Amino acid 62Optimized CD4-CD28 zeta Nucleic acid 63 Optimized CD4 4-1BB zeta Nucleicacid 64 Optimized CD4 extracellular domain Nucleic acid 65 OptimizedCD8α extracellular hinge Nucleic acid 66 Optimized CD8α TM Nucleic acid67 Optimized CD28 TM and ICD Nucleic acid 68 Optimized 4-1BB Nucleicacid 69 Optimized CD3 zeta Nucleic acid 70 Optimized PG9 CD3zeta Nucleicacid 71 Optimized PGT128 CD3zeta Nucleic acid 72 Optimized VRC01 CD3zetaNucleic acid 73 Optimized 3BNC60 CD3zeta Nucleic acid 74 OptimizedPGDM1400 CD3zeta Nucleic acid 75 Optimized PG9 Nucleic acid 76 OptimizedPGT128 Nucleic acid 77 Optimized VRC01 Nucleic acid 78 Optimized 3BNC60Nucleic acid 79 Optimized PGDM1400 Nucleic acidLentivirus Harvesting, Concentration, and Transduction of Primary CD8 TCells:

Plasmid preparations were combined with commercially availablelentiviral packaging plasmids expressing VSV glycoprotein, HIV-1 Gag andPol, and Rev (pRSV.REV, pMDLg/p.RRE, pVSV-G) and transfected ontoHEK293T cells with pTRPE transfer vectors using the Lipofectamine 2000transfection reagent (Invitrogen, Life Technologies). The same processwas completed with the RD114 Env and MMLV gag/pol plasmids (pMSCV RD114and pNGLV3g/p) to generate the retrovirus. 30 ml aliquots of supernatantwere collected at 24 and 48 hour time points and concentrated byultracentrifugation overnight at 8,500RPM at 4° C.. Supernatant wasaspirated and cell pellet was resuspended in 1.2 ml total volume, flashfrozen on dry ice, and transferred to −80° C. for storage.

Cell Culture—Experimentai Protocol:

Healthy donor CD8 and CD4 T lymphocytes were obtained and activated withDynabeads—αCD3/αCD28 coated T-expander beads. T cells were purified bynegative selection using the RosetteSep Human CD4+ or CD8+ T CellEnrichment Cocktails according to the manufacturer's protocols (StemCellTechnologies). T cells were cultured at 1×10⁶ per mL in RPMI 1640 (LifeTechnologies) supplemented (ThermoFisher Scientific) with 10% fetal calfserum (Seradigm), 1% Penn Strep (Life Technologies), 2 mM GlutaMax (LifeTechnologies), and 25 mM HEPES buffer (Life Technologies). T cells werestimulated with anti-CD3/CD28 coated Dynabeads (Life Technologies) at a3:1 bead to cell ratio and 100-300 international units per milliliter ofrecombinant human interleukin 2 for 5 days and then beads were removed.1 day after stimulation, 200 ul of lentivirus supernatant was added to0.5×10⁶ cells. MMLV vector transduction was performed on days 3 and 5,with 1 ml virus supernatant added to a Retronectin (Takara) coated 24well plate and spinoculated according to the manufacturer'sinstructions. Medium was doubled on day 3 and changed completely on day5, and then added every other day throughout cell culture, or asnecessary based on cell counts.

Hiv-1 Infections.

Two days after removing the anti-CD3/CD28 beads, CD4 T cells wereinfected with CCR5-tropic HIV strain Bal, and 24 hours later wereco-cultured at varying effector to target (E:T) ratios with CAR+ CD8 Tcells. The CCR5-tropic HIV-1 Bal viral stock (280 ng/ml p24) wasprepared by harvesting the cell free supernatant from anti CD3/CD28activated CD4 T cells. Activated CD4 T cells were infected by addingapproximately lml of supernatant per 20 million cells 2-3 days afterremoving beads. The following day CD4 and CD8 T cells were co-culturedat varying E:T ratios and HIV spread was monitored by intracellular p24staining was detected with the KC57 anti-Gag-RD1 antibody (BeckmanCoulter) and the Invitrogen Fix and Perm buffers, according themanufacturers' instructions.

In Vitro Cytotoxicity and Cytokine Assays

In vitro killing of K562 cells expressing HIV-1 gp120/41 envelope (YU-2)was tested with a ⁵¹Cr-release assay. 5×10⁵ target cells were loadedwith 50 μCi of Na₂₅₁CrO₄ (Perkin Elmer) for 90-120 minutes, washed twiceand resuspended in phenol red-free medium with 5% FBS. CAR or mocktransduced T cells (8-10 days after initial activation) wereco-incubated with loaded target cells for 4 hours at variouseffector:target (E:T) ratios, and chromium release into the supernatantwas measured with a MicroBeta2 plate counter (Perkin Elmer).Intracellular cytokine production was measured after co-culturing 5×10⁵NTD, CD4 CAR, or scFv CAR transduced T cells at a 1:1 E:T ratio withparental K562s, HLA-DR*0401 transduced K562s, or HIV YU2 Env transducedK562s for 6 hours. Cytokine production was detected as previouslydescribed (86) using rat anti human IL-2 APC (BD biosciences), mouseanti human MIP-1β PerCP Cy5.5 (BD biosciences), mouse anti human IFN-γFITC (BD biosciences), and mouse anti human CD107a PE (BD biosciences),along with Invitrogen Fix and Perm buffers. Spontaneous release bytarget cells (without effector cells) was analyzed in the same volume,and maximum release was assessed by treating target cells with SDS at afinal concentration of 5%. Percent specific lysis=[(ExperimentalRelease−Spontaneous Release)/(Maximum Release−Spontaneous Release)]*100.All experiments were performed with at least 3 replicates, and withprimary human T cells from 3 distinct donors. Interferony production wasquantified by ELISA (R&D) according to the manufacturer'srecommendations in duplicate culture supernatants after co-culture of3×10⁵ effector cells and 10⁵ target cells in 400 ul for 24 h.

CCR5 ZFN Disruption.

Previously described CCR5 specific ZFNs were cloned into a RNAexpression vector (Beatty et al., Cancer immunology research 2, 112-120(2014)). The mMessage mMachine T7 Transcription Kit (Ambion, ThermoFisher Scientific) was utilized to generate capped, in vitro transcribedRNA, which was subsequently purified with the RNeasy Mini Kit (Qiagen)and eluted in RNase-free water at 1 mg/ml. Normal donor CD8 T cells werewashed three times with OPTI-MEM medium and re-suspended at a finalconcentration of 1×10⁸/ml prior to electroporation. 10E6 T cells in 0.1ml were mixed with 30 g of RNA encoding for each ZFN, electroporated ina 2 mm cuvette (Harvard Apparatus BTX) using an ECM830 Electro SquareWave Porator (Harvard Apparatus BTX), and incubated at 30° C. for 48hours prior to activation with αCD3/αCD28 coated Dynabeads (LifeTechnologies).

To measure efficiency of genome modification by CCR5 ZFNs, genomic DNAwas purified from T cells and used to prepare samples for Illumina deepsequencing (Wang et al., Nucleic Acids Res 44, e30 (2016). Briefly, theCCR5 target region was amplified and MiSeq adaptor was added using anested PCR method with the following 2 CCR5-specific primer pairs. CCR5Out-Outl primers: CTGTGCTTCAAGGTCCTTGTCTGC (SEQ ID NO: 80) andCTCTGTCTCCTTCTACAGCCAAGC (SEQ ID NO: 81); CCR5 MiSeq adaptor primers:ACACGACGCTCTTCCGATCTNNNNNGCCAGGTTGAGCAGGTAGATG (SEQ ID NO: 82) andGACGTGTGCTCTTCCGATCTGCTCTACTCACTGGTGTTCATCTTT (SEQ ID NO: 83). Sequencebarcodes were then added in the subsequent PCR reaction using thebarcode primer pairs. For analysis of gene modification levels, acustom-written computer script was used to merge paired-end 150 bpsequences, and adapter trimmed via SeqPrep (John St. John,https://github.com/jstjohn/SeqPrep). Reads were aligned to the wild-typetemplate sequence. Merged reads were filtered using the followingcriteria: the 5′ and 3′ ends (23 bp) must match the expected ampliconexactly, the read must not map to a different locus in the target genomeas determined by Bowtie2 (Yu et al., Journal of virology 81, 1619-1631(2007)) with default settings, and deletions must be <70% of theamplicon size or <70 bp long. Indel events in aligned sequences weredefined as described previously (Gabriel et al., Nat Biotech 29, 816-823(2011)), with the exceptions that indels of lbp in length were alsoconsidered true indels to avoid undercounting real events, and trueindels must include deletions occurring within the sequence spanningbetween the penultimate bases (adjacent to the gap) of the binding sitefor each partner ZFN.

Humanized Mouse Model.

6 week old NSG (NOD-scid IL2Rgnull) mice were obtained from The JacksonLaboratory (JAX) and at 7 weeks treated with 30 mg/kg Busulfan mixed 1:1with PBS. 24 hours later mice were injected via tail vein with 10×10⁶human lymphocytes in 100 ul 0.5% human serum albumin in PBS. Three weekslater mice were infected with 15 ng of the CCR5-tropic Bal strain of HIVmixed 1:1 with PBS via tail vein injection. Peripheral blood wasobtained by retro-orbital bleeding, and human CD4 and CAR+ CD8lymphocyte counts were enumerated using BD lysis buffer and BD TruCounttubes as previously described (Richardson et al., Molecular therapy: thejournal of the American Society of Gene Therapy 22, 1084-1095 (2014)),staining with mouse anti human CD45 PerCp Cy5.5 (BD Biosciences), mouseanti human CD4 BV421 (Biolegend), and mouse anti human CD8α BV711(Biolegend).

HIV Viral Load Assay.

RNA was extracted from 10-30 μl of plasma depending on availabilityusing methods as described in (Cillo et al., J Clin Microbiol 52,3944-3951 (2014)) and reconstituted in a final volume of 15 ul. Prior toextraction, a uniform quantity of Replication Competent Avian Sarcoma(RCAS) virus spiked into each plasma sample and amplified separately toverify virus/RNA recovery and absence of PCR inhibition (Palmer et al.,J Clin Microbiol 41, 4531-4536 (2003)). RNA was reverse transcribedusing random hexamers and quantified by Q-PCR using the LightCycler 480Probes Master (Roche; Indianapolis, Ind.) on an ABI 7500FAST real-timethermocycler using an in vitro transcribed RNA standard. For eachsample, the Q-RT-PCR reaction was run in duplicate on 5 ul RNA;no-reverse transcriptase reaction and RCAS amplification were run on onewell per sample using 2.5 ul RNA. The HIV-1 primer/probe targets the polgene and detects all group M clades as described in (Cillo et al., JClin Microbiol 52, 3944-3951 (2014)), and RCAS amplification usedprimer/probe as described in (Palmer et al., J Clin Microbiol 41,4531-4536 (2003). HIV-1 quantification was normalized to equivalentvolumes of starting plasma.

The results of the experiments are now described.

Example 1: Lentiviral Backbone Augments CD4 CAR Expression and ControlOver HIV Replication

Preclinical studies testing the CD4 CAR used in the previous clinicaltrials showed that T cells expressing this construct had equivalentantiviral activity to naturally generated HIV-specific T cells. Thehypothesis of the failure of these clinical trials to demonstratedurable clinical responses is that the T cells used in these clinicaltrials were not any more potent than the endogenous HIV-specific T cellresponse. The present invention optimizes each component of the CAR in astep-by-step manner to augment the potency by which T cells control HIVreplication.

Primary human CD8 T cells were transduced with the original murineretroviral vector (MMLV) clinical trial construct and with analogousthird generation lentiviral vector construct that also used the PGKpromoter to drive CD4 CAR expression. Lentiviral transduction of primaryhuman CD8 T cells consistently resulted in a ˜10-fold higher medianfluorescence intensity (MFI) of CAR expression compared to murineretrovirus.

The present new, improved CD4 membrane-bound chimeric receptor (CD4 zetaconstruct) with the lentiviral vector, EF1α promoter, and CD8αtransmembrane domain controlled HIV-1 at much lower E:T ratios (untilroughly 1:100) than the constructs with the PGK promoter and CD4transmembrane domains (lose control at 1:5 and 1:10). Even in view ofhigher expression of the lentivirus PGK CD4 TM CAR compared to theretrovirus PGK CD4 TM CAR, a small benefit was seen in terms of controlover HIV-1 replication. Importantly, higher expression of the EF1α CD8TM construct did not promote higher rates of infection, as the CD8sremaining uninfected until approximately the 1:100 ratio whereas the PGKCD4 TM engineered CD8 cells became infected at lower E:T ratios (FIGS.2A-2D). Upon diluting the CD4 CAR transduced CD8 T cells to lower E:Tratios, T cells transduced with the lentiviral vector were superior atcontrolling HIV replication compared to T cells transduced with theretroviral vector (FIGS. 2F-2G). Ultimately neither population oftransduced CD8 T cells could control HIV spread at a 1:50 E: T ratio.Thus the promoter and transmembrane changes of the present inventionimproved the CD4 membrane-bound chimeric receptor efficacy and providedan increased control over HIV-1 infection.

In contrast to recent reports that CD4 CAR transduced CD8 T cells aresusceptible to infection by cell free virus (Liu et al., Journal ofvirology 89, 6685-6694 (2015); Zhen et al., Molecular therapy: thejournal of the American Society of Gene Therapy 23, 1358-1367 (2015)),the present results only show detection intracellular gag in CAR CD8 Tcells after diluting to low E: T ratios with HIV-infected CD4 T cells(FIG. 2E). Relative to non-transduced CD8 T cells, high levels ofintracellular gag were shown to be present in CAR-transduced CD8 Tcells, indicating the CD4 CAR renders these cells susceptible toinfection (FIG. 2E). Importantly, the higher expression of CD4 on thelentivirus transduced CD8 T cells did not promote greater levels ofinfection of these cells or infection at higher E:T ratios. This datademonstrated that lentiviral vectors are the preferred method ofintroducing constructs that re-direct T cells to target HIV.

Example 2: EF1α Promoter and CD8α Transmembrane Domains Improve CARExpression and Control Over HIV-1

The EF1α CD8TM construct was shown to control HIV-1 replication at thelowest E:T ratios. Simply increasing expression of the CD4 TM with theEF1 a promoter (FIGS. 3A-3B, middle column) improved control over HIV-1replication, but the combination of increased expression with the EF1αpromoter and CD8α transmembrane substitution produced the best controlover HIV-1. Furthermore, CD8α TM appeared to reduce infection of the CD4membrane-bound chimeric receptor CD8 cells (FIGS. 3A-3B right set ofcolumns), particularly at the 1:50 ratio for the EF1α constructs and the1:25 ratio for the PGK constructs.

Substitution of the CD8α TM domain decreased infection of CAR+ CD8 Tcells regardless of the promoter used at the 1:25 and 1:50 E:T ratios(FIG. 3B). However, as seen in FIGS. 2A-2G, the CAR+ CD8 T cells can bediluted to the point where they no longer can control HIV infection andsuccumb to infection themselves. Thus, altering the viral vector,promoter, and transmembrane domains afforded a 50-fold increase inpotency over the clinical trial retrovirus, resulting in completecontrol over HIV replication at a 1:50 E: T ratio in vitro (FIGS.3C-3D).

Thus, the CD8α transmembrane change was a critical modification toimprove control of HIV-1 replication.

Example 3: Broadly Neutralizing Antibody Based CARs can Control HIV-1

As seen in FIGS. 4A-4B, both single chain variable fragment (scFv)broadly neutralizing antibody (bnAb)-based CARs and CD4 membrane-boundchimeric receptor can control HIV-1 infection. A panel of scFvs (seetable in FIG. 4A) that ranged in their HIV-1 binding breadth andneutralization potency were cloned into the same EF1α-CD8α TM-zetaconstruct backbone as the CD4 membrane-bound chimeric receptor andassessed for their ability to control HIV-1 replication. Cr51 releasekilling assay from a 4 hour co-culture showed that CD8 T cellsexpressing any of the scFv CARs or the CD4 membrane-bound chimericreceptor, but not non-transduced (NTD) T cells, lysed Env-expressingtarget cells. This indicates the scFv CARs were expressed with properfolding/conformation and able to bind Env (FIG. 4B). Many of the scFvCARs consistently produced high levels of intracellular cytokines inresponse to Env-expressing targets. Both the CD4 membrane-bound chimericreceptor and the scFv CARs demonstrated lysis of Env target cellsrelative to NTD controls up to a E:T ratio of 1:50-1:200 (FIG. 4C),which was superior to the activity of the KF11 TCR shown in FIG. 1,which demonstrated efficacy up to a 1:25 E:T ratio.

Without wishing to be bound by theory, it is understood that use of aCAR that targets HIV, wherein the CAR comprises an anti-HIV scFv will bebeneficial for treatment of subjects that have reservoir populations ofHIV-infected cells. The invention therefore includes such a CAR for suchuse in one aspect of the invention.

Example 4: CD4 CAR is Over 100-Fold More Potent than HIV-Specific EliteController TCR In Vitro

Elite controllers (ECs) are rare individuals who are able to control HIVreplication in the absence of ART. Certain HLA alleles, such as HLA-B57,are overrepresented in elite controller cohorts, suggesting that T cellresponses play a key role in controlling HIV replication in theseindividuals (Walker et al., Nature reviews. Immunology 13, 487-498(2013)). HIV-specific T cells isolated from ECs have higher cytolyticpotential than HIV-specific T cells isolated from HIV progressors(Migueles et al., PNAS 97, 2709-2714 (2000); Saez-Cirion et al., PNAS104, 6776-6781 (2007)). Therefore, to determine whether T cellsexpressing a CD4 CAR are able to control HIV replication better than Tcells expressing a HLA-B57 restricted TCR isolated from an elitecontroller that is associated with better control over HIV replicationin patients, HLA-B57 expressing primary human CD8 T cells weretransduced with a TCR specific for KF11 (HIV p24Gag epitope KAFSPEVIPMF,SEQ ID NO: 42) that was also expressed under the EF1α promoter, andtheir ability to limit HIV spread was determined using CD4 T cells fromthe same donor. KF11 TCR is known in the art to be associated withbetter control over HIV-1 replication in patients and is one of the mostpotent patient-derived TCRs against HIV-1. While KF11 TCR-transduced CD8T cells reduced HIV replication down to a 1:25 E:T ratio, completecontrol over HIV replication was never achieved (FIGS. 1A-1B and FIGS.3A-3D). In contrast, the CD4 CAR of the present invention controlled HIValmost completely down to a 1:100 E: T ratio. These results revealedthat CD4 zeta CAR controls NL4-3 HIV-1 replication better than theHLA-B*57 restricted KF11 elite controller TCR.

These data indicates that CD4 CAR of this invention has much greaterpotency than an endogenous HIV T cell response and suggests that T cellsexpressing optimized CD4 CAR will be able to control HIV replicationafter ART removal at an effector to target ratio that can be achieved byadoptive T cell therapy.

Additionally, FIGS. 5A-5B reveal that CD28 costimulation promotedcontrol over HIV-1 Bal. Another membrane-bound chimeric receptorconstruct that consistently controlled HIV-1 as well or better than CD4zeta construct was CD4 CD28 zeta construct.

Example 5: CD28 and 4-1BB Costimulation have Opposing Effects on theControl of HIV-1 Replication In Vitro

T cells require costimulatory signals for proliferation, effectorfunction, and long-term survival. Additional costimulatory domains, suchas CD28 and 4-1BB, have been incorporated into more recent CAR designs,which have proven necessary for inducing durable CAR T cell responses invivo (Van der Stegen et al., Nature reviews. Drug discovery 14, 499-509(2015)). A panel of CD4 CARs that incorporated a variety ofcostimulatory domains were generated in conjunction with the CD3-zetadomain, including CD28, 4-1BB, CD28+4-1BB, OX40, ICOS, or CD27 and theirability to control HIV infection in vitro was tested. CD8 T cellsexpressing CARs that contained 4-1BB, CD27, or ICOS costimulationdomains did not control HIV as effectively as T cells expressing CARthat only contained CD3-zeta signaling domain, suggesting that thesecostimulatory pathways interfered with T cell control of HIV replication(FIGS. 5A-5B). CARs containing OX40 or a combination of CD28 and 4-1BBcontrolled HIV to similar levels as CARs containing only CD3-zeta. Incontrast, CD28 improved control in vitro, indicating that CD28costimulation may be beneficial in HIV-specific CARs.

Example 5: CD4 CARs Specifically Respond to Env+ Cells and not MHC ClassII+ Cells

Preclinical data demonstrated that T cells expressing the clinical trialCD4 CAR did not kill Raji cells, which express high levels of MHC classII, the low affinity ligand of CD4 (Romeo et al., Cell 64, 1037-1046(1991)). However, the long half-life of CD4 CAR T cells in patients wasspeculated to be a result of low affinity interactions with MHC class IIor possibly the release of Env during viral blips (Scholler et al.,Science translational medicine 4, 132ra153 (2012)). To determine if thepreviously described vector modifications facilitated off-targetreactivity to MHC class II, CD4 CAR+ CD8 T cell responses were measuredagainst a K562 cell line stably expressing high levels of theHLA-DR*0401 allele (see FIG. 14). CD8 T cells were transduced withoptimized CD4 CARs containing CD3-zeta, 4-1BB-CD3-zeta, or CD28-CD3-zetacostimulatory domains and cultured with unmodified K562 target cells,HLA-DR*0401+K562 cells, or HIV YU2 Env+K562 cells. CAR transduced CD8 Tcells produced IL-2, CD107a, IFN-γ, and MIP-1β in response to HIV Env+targets but not in response to HLA-DR+ or parental K562s, with the mostrobust production in CD28-containing CAR T cells (FIG. 11A). A smallamount of MIP-1β signal was observed for all CARs when mixed with eitherparental or HLA-DR expressing targets that was not observed innontransduced controls, likely due to some constitutive, non-antigenspecific signaling observed in CAR expressing T cells. However, comparedto CAR T cells cultured alone or with parental K562s, no additionalcytokine or CD107a production was detected in response to MHC IIexpressing cells.

A co-culture assay was performed to determine if CAR+ CD8 T cells couldkill MHC class II targets, despite the lack of cytokine production. CD4CARs containing the CD28-CD3-zeta costimulatory domains were added to a1:1 mixture of HLA-A*02+/GFP+ K562s and HLA-DR*0401+/mCherry+ K562s andthe ratio of the two K562 cell types was measured over time. Prolongedculture over 72 hours did not result in a reduction of the proportion ofHLA-DR*0401+K562s (FIGS. 11B-11C). These results suggest that use of there-engineered CD4 CAR will be as safe to use in humans as the originalCD4 CAR vector.

Example 6: T Cells Expressing Optimized CD4 CAR Control HIV-1Replication and Expand to Much Greater Levels In Vivo than the FirstGeneration CD4 CAR

CD19-specific CARs containing the CD28 and CD3-zeta signaling domainshad superior in vitro activity than those containing the 4-1BB andCD3-zeta signaling domains, but the 4-1BB containing CARs provedsuperior in humanized mouse models and ultimately in patients with Bcell leukemias ((Porter et al., Science translational medicine 7,303ra139 (2015); Milone et al., Molecular therapy: the journal of theAmerican Society of Gene Therapy 17, 1453-1464 (2009); Brentjens et al.,Blood 118, 4817-4828 (2011)). In order to determine whether the same wastrue for HIV-targeting CARs and if optimized CD4 CARs of this inventioncould control HIV better in vivo then the original CD4 CAR constructthat was previously tested in the clinic, cohorts of mice were infusedwith 10 million human T cells, comprised of 8 million CD4 T cells and 2million CD8 T cells. Four groups of mice were compared with differentCD8 effector cell populations: nontransduced (NTD), transduced with theoptimized lentiviral vector containing either CD28 (28z) or 4-1BBcostimulatory domains (BBz), or transduced with the clinical trialMMLV-based vector (CD4z).

Pre-infection baseline CD4 T cell counts did not differ significantlybetween the NTD, BBz, or 28z groups, and were significantly higher forthe CD4z treated mice (FIG. 12A). Mice were then infected with theCCR5-tropic HIV strain Bal, and after 22 days of infection, endpointperipheral CD4 T cell counts were enumerated. Mice infused with T cellsexpressing the BBz or 28z construct showed a 17-fold and 177-foldexpansion of the number of human CD4 T cells, respectively (FIG. 12B).In contrast, peripheral CD4 counts remained very low in mice treatedwith nontransduced CD8 T cells or CD4z T cells, presumably due toHIV-mediated depletion (FIG. 12B). Examination of the number of CD4 CAR+CD8 T cells in the different mouse cohorts revealed 389-fold, 587-fold,and 2-fold expansions in the BBz, 28z, and CD4z T cells, respectively(FIGS. 12C-12D). The ability of the CD4 CAR T cells to control viralload were also examined in this model. Seven days following HIVinfection, BBz CAR T cells exhibited the greatest control over virusreplication, with mostly undetectable virus loads, whereas plasma fromNTD, 28z, and CD4z treated animals contained approximately 1 copy of HIVRNA per μl (FIG. 12E). 18 days post infection, the median copy number ofHIV RNA was reduced by more than 10 fold in BBz and 28z treated animals,compared to the mice that were treated with NTD T cells (FIG. 12F).However, CD4z treated mice did not statistically reduce HIV RNA comparedto NTD treated mice. While either optimized CD4 CAR controlled

HIV better than the clinical trial CAR, these results indicate thatdifferent costimulatory domains have disparate protective roles in HIVinfection, with CD28 protecting CD4 T cells to the greatest extent and4-1BB promoting early control over HIV replication.

Example 7: CCR5-ZFN Modified CD4 CAR CD8 T Cells are Enriched In Vivo

Despite increased control over HIV replication in vitro and in vivo, thein vitro data suggested that transduction of CD8 T cells withHIV-specific CARs rendered them susceptible to infection. To determinewhether these CAR T cells become infected in vivo, CAR T cells resistantto infection enrich were tested in the presence of HIV in vivo. Thesurvival benefits afforded by gene editing the CCR5 HIV co-receptorusing zinc finger nucleases (ZFNs) has previously been demonstrated inprimary human CD4 T cells (Perez et al., Nat Biotechnol 26, 808-816(2008); Tebas et al., Nature reviews. Immunology 13, 693-701 (2013)).However, similar enrichment analyses on CD4 CAR+ CD8 T cells in thepresence of HIV infection had not been explored. To determine ifco-receptor editing promotes better enrichment of CAR+ CD8 T cells as ameans to determine with CD4 CAR CD8 T cells are susceptible to infectionin vivo, four additional groups were included in the experimentdescribed in FIGS. 12A-12F in which the CD8 T cells were treated withCCR5 ZFNs and subsequently transduced with each of the CD4 CARconstructs.

CCR5 disrupted alleles were enriched in mice that were treated with CD4CAR T cells and infected with HIV relative to non-transduced CD8 T cells(FIG. 13A). HIV-infected mice engrafted with non-transduced CD8 T cellshad a median disruption frequency of 0.4%. As these CD8 T cells were notCD4 CAR transduced, they were not expected to enrich in the presence ofHIV. Mice treated with BBz, 28z, or CD4z T cells had median disruptionfrequencies of 14.4%, 29.6%, and 9.5%, respectively, indicating thatHIV-resistant, CD4 CAR-expressing CD8 T cells enrich in the presence ofHIV infection. CCR5 disruption enrichment is influenced by the abilityof CAR+ CD8 T cells to expand in the presence of HIV, as shown in FIG.13B and FIG. 15, and thus greater antigen-specific expansion of 28z CART cells could be responsible for the greater CCR5 enrichment seen inFIG. 8A. This data also shows that BBz engineered T cells are able topersist better than 28z engineered T cells in the absence of antigen,which is consistent with findings using tumor specific CARs (Milone etal., Molecular therapy: the journal of the American Society of GeneTherapy 17, 1453-1464 (2009); Carpenito et al., PNAS 106, 3360-3365(2009)). Together, this data suggest that HIV infection of HIV-specificCAR T cells does occur, and strategies that protect these cells from HIVinfection will likely be necessary to ensure durable control of HIVinfection.

It was also examined whether rendering CD4 CAR CD8 T cells resistant toHIV infection enhanced their ability to control HIV-1 infection. After22 days of infection, in general there were no significant differencesin peripheral blood CD4 T cell counts or expansion of CAR+CD8 T cells ininfected mice given ZFN-treated cells or non-ZFN treated cells, with theexception of lower endpoint CD4 T cell counts in 28z mice treated withZFNs compared to non-ZFN treated (FIGS. 16A-16B). However, there was atrend for greater CD4 and CAR+ CD8 T cell expansions in ZFN-treated,CD4z mice (FIGS. 16A-16B). In 28z or BBz mice, which had low viral loadsin the absence of ZFN treatment, no further decrease in viral RNAresulted from ZFN treatment (FIG. 13C). On the other hand, a significantdecrease in plasma viral RNA was seen in mice given ZFN-treated CD4z Tcells, to the point where plasma virus was no longer significantlyhigher than mice given the BBz or 28z CARs. As the viral loads wereapproximately a log-fold higher in the CD4z retrovirus treated micewithout ZFNs compared to BBz or 28z treated mice without ZFNs, it iseasier to detect a significant reduction in viral load in the context ofpoorly controlled virus replication resulting from ZFN treatment.Moreover, the short duration of the experiment could have precludedseeing full benefit of ZFN treatment on CAR T cells, including adetectable enrichment of transduced CD8 T cells, particularly in theCD4z treated mice in which they yielded the most antiviral benefit. Insummary, in the absence of ZFN treatment, optimized CARs are superior tothe clinical trial CD4z in vivo; however, if the T cells expressing theoriginal CAR construct can be protected by CCR5 disruption, then controlover HIV replication can be achieved within this relatively short invivo assay.

Overall, the improved control of HIV replication disclosed herein isimpressive (FIGS. 17A-17C). This invention includes several improvementsto the previously disclosed CD4 membrane-bound chimeric receptor thatresult in a 50-fold increase in the ability of killing HIV-1 infectedcells. This increased efficacy, unlike the 1st generation CD4membrane-bound chimeric receptor, titrates to effector:target (E:T)ratios that are physiologically relevant in in vitro assays, suggestingthat these improved CD4 membrane-bound chimeric receptor constructs willlikely be more effective in reducing viral loads in patients.

One of the major challenges for long-term control of HIV infection isthe immune escape of some virus mutants. The scFv based CARs (scFv-CARs)of the present invention address this challenge and were shown to bebeneficial for treatment of subjects that have reservoir populations ofHIV-infected cells. The addition of T cells expressing scFv based CARsmay further augment the ability of engineered T cells to provide durablecontrol of HIV replication in the absence of ART. As seen in FIG. 9A andFIG. 10A, VRC01, VRC01c and 3BNC60-CARs showed specific lysis of K562cells expressing HIV-1 envelope gp120/41 (YU-2 isolate) but not of K562cells expressing CD19 which confirmed that scFv-CARs cytotoxicity isboth potent and specific. Also, interferon gamma (IFNg) released byscFv-CAR T cells was tightly correlated with cells' killing efficacy(FIG. 9C and FIG. 10C). Additionally, scFv-CARs cytotoxicity wasincreased further by using a killer cell immunoglobulin-like receptor(KIR) cytoplasmic domain.

The present invention provides an enormous potential for improved HIVtherapy. T cells isolated from HIV patients can be transduced with thescFv-CARs and/or CD4 membrane-bound chimeric receptors, expanded ex vivoand reinfused into the subject. The transduced T cells recognize gp120expressing cells and become activated, resulting in killing of HIVharboring cells. This approach can be applied to any virus mediateddisease in which a target antigen may shift or drift and thereforeescape immunosurveillance by normal antibodies (e.g. hemagglutinin ofinfluenza A). This therapy can be combined with other known antibodytherapies, HAART or may be combined with viral inducers during or beforeCAR therapy.

Prophylactic application of the present invention is also possible inhigh risk populations, since the T cells should kill infected CD4 cells,which prevents establishment of a HIV repertoire.

Other Embodiments

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. An isolated nucleic acid sequence encoding amembrane-bound chimeric receptor comprising a CD4 extracellular domain,a CD8alpha hinge, a CD8alpha transmembrane domain, and a signalingdomain comprising a 4-1BB costimulatory signaling region, wherein theCD4 extracellular domain is encoded by a nucleic acid sequencecomprising SEQ ID NO: 3 or 64 and is capable of recognizing and bindinga HIV infected cell.
 2. The isolated nucleic acid sequence of claim 1,wherein the CD8alpha hinge is encoded by a nucleic acid sequencecomprising SEQ ID NO: 4 or
 65. 3. The isolated nucleic acid sequence ofclaim 1, wherein the signaling domain further comprises a CD3zetasignaling domain encoded by a nucleic acid sequence comprising SEQ IDNO: 6 or
 69. 4. The isolated nucleic acid sequence of claim 1, whereinthe CD4 extracellular domain specifically binds to the HIV envelope(Env) glycoprotein.
 5. A vector comprising the isolated nucleic acidsequence of claim
 1. 6. The vector of claim 5, wherein the nucleic acidsequence comprises at least one from the group consisting of SEQ ID NOs:3-6, 63-66, 68, and
 69. 7. An isolated amino acid sequence encoding amembrane-bound chimeric receptor comprising a CD4 extracellular domain,a CD8alpha hinge, a CD8alpha transmembrane domain, and a signalingdomain comprising a 4-1BB costimulatory signaling region, wherein theCD4 extracellular domain is encoded by a nucleic acid sequencecomprising SEQ ID NO: 3 or 64 and is capable of recognizing and bindingan HIV infected cell.
 8. The isolated amino acid sequence of claim 7,wherein the CD4 extracellular domain comprises the amino acid sequenceSEQ ID NO:
 46. 9. The isolated amino acid sequence of claim 7, whereinthe CD8alpha hinge comprises the amino acid sequence SEQ ID NO: 47, andthe CD8alpha transmembrane domain comprises the amino acid sequence SEQID NO:
 48. 10. The isolated amino acid sequence of claim 7, wherein thesignaling domain further comprises a CD3zeta signaling domain comprisingSEQ ID NO:
 51. 11. The isolated amino acid sequence of claim 7, whereinthe 4-1BB costimulatory signaling region comprises SEQ ID NO:
 50. 12.The isolated amino acid sequence of claim 7, wherein the CD4extracellular domain specifically binds to the HIV envelope (Env)glycoprotein.
 13. A vector comprising a nucleic acid encoding amembrane-bound chimeric receptor comprising a CD4 extracellular domain,a CD8alpha hinge, a CD8alpha transmembrane domain, and a signalingdomain comprising a 4-1BB costimulatory signaling region, wherein theCD4 extracellular domain is capable of recognizing and binding an HIVinfected cell, wherein the nucleic acid sequence encodes the amino acidsequence SEQ ID NO:
 45. 14. The vector of claim 13, wherein the vectorcomprises an EFa promoter.
 15. A modified cell comprising the isolatednucleic acid sequence of claim
 1. 16. The modified cell of claim 15,wherein the cell is selected from the group consisting of a T cell, anatural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and aregulatory T cell.
 17. The modified cell of claim 16, wherein thenucleic acid sequence is selected from the group consisting of a DNA andan mRNA.
 18. The modified cell of claim 16, wherein the nucleic acidsequence is introduced into the cell by at least one procedure selectedfrom the group consisting of electroporation, usage of a lentivirus,usage of a retrovirus and a chemical-based transfection.
 19. Acomposition comprising the modified cell of claim
 16. 20. Apharmaceutical composition comprising the modified cell of claim 16 anda pharmaceutically acceptable carrier.
 21. A method for stimulating acellular immune response in a HIV infected mammal, the method comprisingadministering to the mammal an effective amount of the modified cell ofclaim
 16. 22. A method of treating a HIV infected mammal, the methodcomprising administering to the mammal the modified cell of claim 16.23. The method of claim 22, wherein the modified cell is autologous tothe mammal.
 24. The method of claim 23, further comprising administeringantiretroviral therapy (HAART) the mammal.
 25. The method of claim 23,wherein the modified cell and the HAART are co-administered to themammal.
 26. The isolated nucleic acid sequence of claim 1, wherein theCD8alpha transmembrane domain is encoded by a nucleic acid sequencecomprising SEQ ID NO: 5 or
 66. 27. The isolated nucleic acid sequence ofclaim 1, wherein the 4-1BB costimulatory signaling region is encoded bya nucleic acid sequence comprising SEQ ID NO: 68.